Genes involved in astaxanthin biosynthesis

ABSTRACT

Provided herein are nucleic acid, host cell, and polypeptide compositions encoded by the unicellular green alga  Chromochloris zofingiensis , methods of making such compositions, and method of using the compositions to produce astaxanthin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional Application No. 62/471,887, filed Mar. 15, 2017, which application is herein incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. The unicellular green alga Chromochloris zofingiensis produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin; however, much remains unknown about the genome and regulation of metabolism in this alga

C. zofingiensis (division Chlorophyta, class Chlorophyceae, order Sphaeropleales) is a simple ˜4 μm, unicellular, haploid, coccoid alga containing multiple mitochondria, which are visualized typically as a tubular network, and a single interconnected chloroplast that occupies ˜40% of the cell volume and contains starch granules. Most of the mitochondria are in close association with either the nucleus or the chloroplast. However, neither flagella (cilia) nor pyrenoids were visually observed. Because of the lack of obvious morphological characteristics, C. zofingiensis was originally described as a Chlorella species (6), at times transferred to the genera Muriella and Mychonastes, and finally placed using molecular sequencing into the genus Chromochloris (7). Similar to its close relative, the model alga Chlamydomonas reinhardtii, C. zofingiensis exhibits multiple fission with temporal separation between cell growth and cell division. C. zofingiensis primarily divides into two or four daughter cells, but also can divide into 16, 32, or 64 cells (6). The regulation of cell division timing is unknown, but the daughter cells are the same size. Also like C. reinhardtii (8), the nucleus in C. zofingiensis divides prior to chloroplast division. Intriguingly, C. zofingiensis has an extremely high photoprotective capacity compared to other algae and plants (9). Moreover, under specific conditions, C. zofingiensis can dramatically increase the production of lipids and secondary carotenoids (3-5, 10). This alga produces triacylglycerols (TAGs), the preferred lipid precursor for biofuel products and accumulates these to some of the highest levels out of 96 microalgae analyzed (3). Thus, C. zofingiensis is presently considered one of the most promising biofuel feedstocks for commercial production.

Increased production of the highly valuable ketocarotenoid astaxanthin occurs in concert with accumulation of TAGs (4, 5). Astaxanthin has a broad range of commercial applications, including pharmaceuticals, nutraceuticals, cosmetics, food, and feed (11-13). Recent studies have highlighted the antioxidant and anti-inflammatory benefits of astaxanthin for applications in human health including cancer, cardiovascular disease, neurodegenerative disease, inflammatory disease, diabetes, and obesity treatments (11, 12). Although astaxanthin can be produced synthetically, naturally produced astaxanthin is distinct in its esterification and stereochemistry (13-15). These differences result in natural astaxanthin having >20-fold stronger antioxidant activity than synthetic astaxanthin, and only natural astaxanthin has been approved for human consumption (14). Because C. zofingiensis is fast growing, can be cultured under many conditions (including with wastewater), and reaches high culture densities, C. zofingiensis has higher potential to meet worldwide demand than other natural sources, such as the microalga Haematococcus pluvialis, yeast, transgenic plants, and crustaceans (13, 15-17). Thus, C. zofingiensis is a prime candidate to supply the world with natural astaxanthin as well as a source of renewable biofuel. However, improvements to maximize productivity and yield are needed, and key aspects of astaxanthin biosynthesis and regulation remain to be elucidated.

BRIEF SUMMARY OF THE INVENTION

We sequenced and assembled C. zofingiensis nuclear, mitochondrial, and plastid genomes using a hybrid approach, constructed a transcriptome from 14 diverse conditions, examined transcriptomic changes through a shift from normal growth to that in high light, generated and analyzed astaxanthin-deficient mutants, and identified candidate genes involved in algal astaxanthin biosynthesis. The high-quality, chromosome-level genome assembly and accompanying transcriptome, combined with the capacity for genetic transformation (18), establish a molecular foundation to facilitate commercial development of C. zofingiensis.

Astaxanthin is an important and valuable algal bioproduct. In microalgae, astaxanthin is often produced in high abundance under stressful conditions, consistent with the hypothesis that it confers protection against oxidative stress. However, astaxanthin is not coupled functionally or structurally to the photosynthetic apparatus. Instead, astaxanthin functions as an internal sunscreen and antioxidant by absorbing excess light and quenching reactive oxygen species, Additionally, astaxanthin accumulates in cytoplasmic lipid droplets where it could prevent peroxidation of fatty acids.

In one aspect, provided herein are polynucleotides and polypeptides that are participate in the astaxanthin pathway in microalgae; and host cells, including microalgae, plant, yeast, or other host cells, engineered to express such gene to provide for astaxanthin production and/or enhanced astaxanthin production.

Examples of host cells that can be engineered to express an astaxanthin polypeptide of the present invention include bacteria, yeast, fungi, plants, microalgae, cyanobacteria, and the like. Examples include microalgae and cyanobacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Chromochloris zofingiensis nuclear genome. The assembled sequence of the 19 chromosomes of the nuclear genome is shown (top bar in each pair) with the matching chromosomes from the optical map (bottom bar in each pair). Nominal plus strands run 5′ to 3′ left to right. Thin vertical divisions mark BamHI restriction sites (in silico in top bars, optical consensus in bottom bars). Lines from restriction sites on one bar to another indicate a maximally-scoring alignment computed with a dynamic programming algorithm similar to that used in OpGen, Inc.'s MapSolver software. Black squares at chromosome edges indicate sequence assembly has reached telomere-associated repeats. Thick horizontal orange bars indicate explicit assembly gaps (runs of Ns). Thick horizontal yellow bars indicate additional known assembly issues as cataloged in S1 File S4. Light blue background shading shows where alignments are not one-to-one; shading is light green otherwise. Red dots mark possible (peri)centromeric loci. Optical assembly did not reach the end of chromosome 5, and the large sequence gap at the end of chromosome 13 likely begins with ˜24× copies of the rDNA unit. Unplaced contigs/scaffolds and 24 copies of the rDNA unit are shown near the bottom right.

FIG. 2A-C. Gene families. Using a procedure based on reciprocal near-best global amino acid alignments, protein-coding gene families among the six organisms of Table 1 were formed. (FIG. 2A) Phylogram estimated from restriction to putative 1:1:1:1:1:1 orthologs is consistent with existing literature (27). (FIG. 2B) A proportional Venn diagram showing the partitioning of the 15,274 Chromochloris zofingiensis genes by the combination of presence and absence of Monoraphidium neglectum, Chlamydomonas reinhardtii, and Arabidopsis thaliana genes in the families to which the genes belong also suggests these three organisms in this order are increasingly distant from C. zofingiensis. (FIG. 2C) Scatterplots show scrambled syntenic blocks of conserved genes in the algal lineage (similar to FIG. 2, reference 23). (Organism pairs involving the highly fragmented assembly of M. neglectum are omitted.) Each plot uses those gene families that, for the two organisms selected, have exactly one primary gene a in the first organism and exactly one primary gene b in the second organism. A dot with x-, y-coordinates at the midpoints of the span of the coding sequences for a, b is drawn in red if a and b are on the same nominal genomic strands and in green if they are on opposite strands; dots are plotted in a randomized order. Order of assembly sequences (but not nucleotides within sequences) is permuted on both axes so as to compact and emphasize statistically enriched regions (indicated by orange background shading); small numbers running along edges of inside plot frames give relevant portions of the assembly's sequence names for sequences at least 0.5 Mbp in length. Rightward and downward are 5′ to 3′ on assembly plus strands and light gray lines mark assembly sequence boundaries. Further details are given in SI Text of the SI Appendix.

FIG. 3A-B. Chromochloris zofingiensis astaxanthin-deficient mutants. Astaxanthin-deficient mutants were generated using forward genetics; mutations were identified in BKT1. (FIG. 3A) HPLC traces of wild type, bkt1-1, bkt1-2, and bkt1-3 grown under high light (HL, 400-450 μmol photons m's′), showing the mutants' lack of astaxanthin production. Pigment abbreviations are as follows: N (neoxanthin), V (violaxanthin), Ast (astaxanthin), A (antheraxanthin), L (lutein), Z (zeaxanthin), Chl b (chlorophyll b) Chl a (chlorophyll a), α (α-carotene) and β (β-carotene). Pigments were detected at 445 nm with reference at 550 nm (SI Appendix, SI Text). Inset shows high light WT growth with astaxanthin resulting in orange-brown color from astaxanthin (orange) and chlorophylls (green), whereas mutants bkt1-1, bkt1-2, and bkt1-3 do not produce astaxanthin and remain green. Under medium light (ML, 100 μmol photons m⁻²s⁻¹), WT does not produce high amounts of astaxanthin and remains green with similar color as bkt1-1, bkt1-2, and bkt1-3. (FIG. 3B) Pigment levels (mean±SD, N=3 or 4) in HL-grown WT, bkt1-1, bkt1-2, and bkt1-3 showing higher levels of carotenoids with similar levels of chlorophyll. * p<0.05, ** p<0.01, *** p<0.001 (see SI Appendix, SI Text).

FIG. 4. Chromochloris zofingiensis RNA expression during transition to high light. Cultures of C. zofingiensis were grown diurnally (16 h light, 8 h dark) in 100 μmol photons m⁻²s⁻¹ medium light (ML). At t=0, cultures were transferred to 400 μmol photons m⁻²s⁻¹ high light (HL). Samples were collected in quadruplicate at 0, 0.5, 1, 3, 6, and 12 h for ML cultures and at 0.5, 1, 3, 6, and 12 h for HL. Transcript abundances for each sample were determined by RNA-Seq. (A) Principal component analysis (PCA) of the regularized log₂-transformed counts for all 44 samples. The two most significant components, accounting for 95% of variation, are shown. ML (triangles) and HL (circles) are displayed with time point indicated by color. (B) Differentially expressed genes during transition to HL. Expression fold change in HL versus ML was determined for each time point for all genes. Genes at least two-fold up-regulated or two-fold down-regulated are indicated by the height of the bar above or below the line, respectively (p<0.01); the total is indicated above each bar. The regularized log₂-transformed fold change between HL and ML is shown in the black box for each time point as indicated by color. For comparison, the fold change of each of these genes at the other time points is presented flanking the black box. The number of differentially-expressed genes in common between 0.5 and 1 h and between 0.5 and 12 h is indicated by square brackets.

FIG. 5. Chromochloris zofingiensis RNA-Seq expression of select genes during the transition to high light. RNA-Seq was performed on cultures following a shift from medium light (ML, 100 μmol photons m⁻²s⁻¹) to high light (HL, 400-450 μmol photons m⁻²s⁻¹) as described in FIG. 4. C. zofingiensis genes potentially involved in carotenoid biosynthesis, non-photochemical quenching (NPQ), and chlorophyll biosynthesis and degradation were identified by manual curation. On the left, the regularized log 2-transformed fold-change between HL and ML for each of these genes at each time point is plotted as a heatmap. On the right, the regularized log₂-transformed fold-change between each time point relative to t=0 is plotted. Significantly differential genes that are over two-fold up- or down-regulated are indicated by an asterisk and bold text (p<0.01).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the identification of genes in C. zofingiensis that are involved in astaxanthin production.

An “expression vector” or “expression cassette” is a nucleic acid construct, generated recombinantly by genetic engineering technolog or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter. An “expression cassette” may also include embodiments in which a polynucleotide encoding a polypeptide of interest, such as an astaxanthin production protein is integrated into host DNA an a non-native position or is integrated into host DNA such that production of the protein is controlled by a heterologous promoter.

By “host cell” is meant a cell that is genetically modified to contain an exogenous nucleic acid that has been introduced into the host cell by recombinant DNA technology, e.g., an expression vector and supports the replication or expression of the expression vector. The term includes the progeny of the host cell that was initially genetically modified and thus includes the primary transformed cell and progeny derived therefrom without regard to the number of passages. Host cells may be prokaryotic cells including but not limited to, algae, including microalgae, plants, cyanobacteria, or eukaryotic cells including but not limited to, algae, yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo. molecule(s) present at one or more locations in a host cell.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids. The use of the term “peptide or peptidomimetic” in the current application merely emphasizes that peptides comprising naturally occurring amino acids as well as modified amino acids are contemplated.

Any “gene” is meant to refer to the polynucleotide sequence that encodes a protein, i.e., after transcription and translation of the gene a protein is expressed. As understood in the art, there are naturally occurring polymorphisms for many gene sequences. Genes that are naturally occurring allelic variations for the purposes of this invention are those genes encoded by the same genetic locus.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.

The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences (or two or more nucleic acids), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same e.g., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over a specified region (such as the first 100 amino acids of SEQ ID NOS: 1-7), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters are typically used.

The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, polypeptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also encompasses “conservatively modified variants” thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ai, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ai, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms “wild type”, “native”, and “naturally occurring” with respect to an astanxanthin-production protein are used herein to refer to a protein that participated in astanxanthin production, e.g., having an amino acid sequence of any one of SEQ ID NOS:1-7 that has a sequence that occurs in nature.

In the context of this invention, the term “mutant” with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with “variant”. A “non-naturally” occurring protein refers to a variant or mutant polypeptide that is not present in a cell in nature and that is produced by genetic modification, e.g., using genetic engineering technology or mutagenesis techniques, of a native polynucleotide or polypeptide. A “variant” includes any protein comprising at least one amino acid mutation with respect to wild type. Mutations may include substitutions, insertions, and deletions.

An “endogenous” protein or “endogenous” nucleic acid” is also referred to as a “native” protein or nucleic acid that is found in a cell or organism in nature.

A polynucleotide or polypeptide is “heterologous” to an organism or a second polynucleotide or polypeptide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a “heterologous” sequence includes a native astaxanthin production protein having one or more mutations relative to the native amino acid sequence; or a native protein that is expressed in a host cell in which it does not naturally occur. In some embodiments, expression of an astaxanthin production polypeptide by a genetically modified host cell in accordance with the invention is under the control of a light-inducible promoter, e.g., a light-inducible promoter from a microlage or from another species, e.g., cyanobacteria, such as psb2 promoter.

In some embodiments, an “astaxanthin production protein” refers to a polypeptide that functions in astaxanthin production and has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence of any one of SEQ ID NOS:1-7.

The terms “increased expression” and “overexpression” of an astaxanthin production polypeptide are used interchangeably herein to refer to an increase in the amount of polypeptide in a genetically modified cell, e.g., a cell into which an expression construct encoding an astaxanthin polypeptide has been introduced, compared to the amount of polypeptide in a counterpart cell that does not have the genetic modification, i.e., a cell of the same strain or organism without the modification, such as a wildtype host cell. An increased level of expression for purposes of this application is at least 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, compared to the counterpart unmodified cell. The unmodified counterpart cell need not natively express the astaxanthin production polypeptide. Thus, the term “overexpression” also includes embodiments in which the polypeptide is expressed in a host cell that does not natively express the polypeptide. Increased expression can be assessed by any number of assays, including, but not limited to, measuring the level of RNA, the level of CVDE polypeptide, and/or the level of polypeptide activity. Illustrative assays are provided in the Examples section. “Overexpression” in the context of protein activity includes overexpression relative to enodogenous activity such that the overall level of activity in the host cell is increased in the genetically modified host cell.

In some embodiments, a polynucleotide that encodes an astaxanthin production polypeptide as described herein, e.g., that has at least 70% identity, at least 75% identity, at least 80%, at leat 85%, at least 90%, or at leat 95% identity to any one of SEQ ID NOS:1-7; comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOS:15-21. In seom embodiments, the polynucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOS:8-14. In some embodiments, the nucleic acid sequence is codon-optimized for expresson in the host cell.

Expression constructs encoding an astaxanthin production polypeptide as provided by the present disclosure can be prepared using any method. For example, a DNA sequence encoding a astaxanthin protein, can be combined with transcriptional and other regulatory sequences which will direct the transcription of the sequence from the gene in the intended cells, e.g., C. zofingiensis cells. In some embodiments, an expression vector that comprises an expression cassette that comprises the nucleic acid sequence endogen the astaxantin production protein further comprises a promoter operably linked to the nucleic acid sequence. In other embodiments, a promoter and/or other regulatory elements that direct transcription of the astaxanthin nucleic acid sequence are endogenous to the host cell or organism, and an expression cassette comprising the astaxanthin gene is introduced, e.g., by homologous recombination, such that the astaxanthin gene is operably linked to an endogenous promoter and is expression driven by the endogenous promoter.

In some embodiments, the promoter may be from a gene associated with photosynthesis or lipid production in the species to be transformed or another species. For example such a promoter from one species may be used to direct expression of a protein in transformed algae cells. Suitable promoters may be isolated from or synthesized based on known sequences from other photosynthetic organisms.

In some embodiments a promoter may be a constitutive promoter. In some embodiments the promoter is an inducible promoter. In some embodiments, a promoter can be used to direct expression of axtaxanthin nucleic acids under the influence of changing environmental conditions.

In some embodiments, the host cell is an algal host cell, e.g., a green algae host cell, such as unicellular green algal host cell. In some embodiments, the host cell is a C. zofingiensis host cell.

EXAMPLES Whole-Genome Sequencing, Assembly, and Global Architecture.

For whole-genome sequencing and chromosome-level assembly of C. zofingiensis (strain SAG 211-14), we used a hybrid approach blending short reads (Illumina), long reads (Pacific Biosciences of California, Inc.) and whole-genome optical mapping (OpGen, Inc.). The combined power of these multiple approaches yielded a high-quality haploid nuclear genome of C. zofingiensis of ˜58 Mbp distributed over 19 chromosomes (FIG. 1). About 99% of reads from the Illumina genomic libraries were accounted for, and non-placeholder chromosomal sequence covers ˜94% of the optical map. Because genome assembly methods used were not automated, details of the procedure are described in the SI Text of the SI section below.

Genome features of C. zofingiensis were compared to four other green algae: C. reinhardtii, Coccomyxa subellipsoidea C-169, Chlorella sp. NC64A, and Monoraphidium neglectum (the closest relative with a sequenced genome), and the model plant Arabidopsis thaliana (Table 1, SI section text). Similar to most green algae, C. zofingiensis has a genome that is approximately half the size of A. thaliana and C. reinhardtii; yet C. zofingiensis and all known algal genomes have more than double the number of chromosomes of A. thaliana. However, C. zofingiensis has the most balanced G+C content (both for the nuclear genome and just coding sequence) of the six organisms (˜51% and 53%, respectively); while C. subelhpsoidea C-169 is similar to C. zofingiensis, the other algal genomes have high G+C content, and A. thaliana has low G+C content. High G+C content is associated with more fragmentary assemblies. C. sp. NC64A has large number of regions with distinct G+C content, but C. zofingiensis does not. The relative repetitive content of the C. zofingiensis genome, like C. subellipsoidea C-169, appears to be low (≈6%); in contrast, the M. neglectum genome has ≈50% higher and C. sp. NC64A≈100% higher relative repetitive content despite comparable genome sizes among these four algae. The large genomes of C. reinhardtii and A. thaliana have roughly double the relative repetitive content compared to the highest of the other four. After C. subelhpsoidea C-169, C. zofingiensis contains the most repetitive fraction from novel repeats not known in Repbase Update (19), which presently focuses on A. thaliana and C. reinhardtii. In C. zofingiensis, gene density is quite uniform over chromosomes, and there are no grand scale gradients in genes or repeats as found in, for example, A. thaliana where each chromosome has megabasepairs of pericentromeric heterochromatin (20). However, some smaller scale gradients in repeats are found near large assembly gaps and putative (peri)centromeres. RepeatMasker in conjunction with RepeatModeler and Repbase finds ˜5.0% of C. zofingiensis sequence consists of interspersed repeats (˜2.0% LINEs, ˜1.5% LTRs, ˜1.2% unclassified, and ˜0.4% DNA elements) with the remainder mostly simple repeats (˜1.0%) and with some satellites, low complexity sequence, and small RNA (total ˜0.1%).

Complete (circular with no gaps or IUPAC ambiguities) mitochondrial and chloroplast genomes for C. zofingiensis strain UTEX 56 (formerly Bracteacoccus cinnabarinus) were already available as NCBI accessions KJ806268.1 (21) and KT199251.1 (22), respectively. We independently assembled equivalent complete genomes de novo for strain SAG 211-14 (Table 1, SI section). These two strains were isolated from similar habitats in localities ˜300 km apart by different people in sequential years. For the mitochondrial genome, the SAG and UTEX strains were resolved as 41,733 bp and 44,840 bp, respectively, with the same major protein-coding genes, tRNAs, and rRNAs in the same order (SI section) (21). However, a pairwise alignment exhibited only ˜66% nucleotide identity, with divergence concentrated intergenically and in rrnL4 where splicing differs. Restricted to coding sequence, nucleotide identity rises to ˜98%, and amino acid identity is ˜99% in translations under the NCBI Scenedesmus obliquus mitochondrial genetic code. For the chloroplast genome, the SAG and UTEX strains resolved as 181,058 bp and 188,935 bp, respectively, with a ˜6.7 kbp and ˜6.4 kbp, respectively, rRNA-related inverted repeat (SI section) (22). Neither the Illumina short reads nor Pacific Biosciences long reads were able to resolve the relative strand orientation of the two single copy regions for the SAG strain; a single contig was constructed with an arbitrary relative orientation which is opposite that given for the UTEX strain. Again, between the strains, all major protein-coding genes, tRNAs, and rRNAs are the same in the same order. I n comparisons between strains, the single copy regions were reoriented to agree. Nucleotide identity was estimated at ˜83%, with divergence concentrated intergenically and with the largest single difference being a loss in the SAG strain of almost all of a ˜9.3 kbp UTEX region annotated as containing a ptz-like ORF. Coding sequence identity is ˜98%, and translation under the NCBI bacterial, archaeal, and plant plastid code gives ˜97% amino acid identity with lower identity in larger proteins (e.g., FtsH, RpoC2, and Ycf1).

The current C. zofingiensis assembly successfully extended into telomere-associated repeats for 25 of 38(=19+19) chromosome tips, and unplaced contigs may represent another 11 tips leaving only two tips unaccounted. The C. zofingiensis canonical unit appears to be (CCCTAAA)_(n) at 5′ ends (and the reverse complement, (TTTAGGG)_(n), at 3′ ends), similar to C. subelhpsoidea C-169 and C. sp. NC64A and likely M. neglectum, although C. reinhardtii may prefer (CCCTAAAA)_(n). A comparison of counts of apparently telomere-associated reads vs. generic nuclear reads (and constraints imposed by the optical map) suggested an average of ≈3.5 kbp of repeats per chromosome tip.

Based on experience with particularly difficult sequence during assembly phases and analysis of the chromosomal distributions of specific dispersed and tandem repeat families, one region per chromosome was identified as a putative (peri)centromeric locus in most chromosomes. These loci are complex nested insertions of a ˜4.7 kbp circular consensus sequence that consists of ˜4 kbp coding sequence of a Type I/Copia LTR retrotransposon together with a ˜0.7 kbp spacer, as well as some 5S rDNA sequence (but apparently no large tandem arrays of a relatively short unit, such as in A. thaliana); the best NCBI BLASTX hits are to the filamentous green alga Klebsormidium flaccidum and the colonial green alga Volvox carteri. In the current C. zofingiensis version 5 assembly, 39 unplaced assembly sequences contain homology to the consensus unit. These regions are reminiscent of the Zepp clusters described in C. subellipsoidea C-169 (23), although the Zepp element is LINE-like and not of LTR type. Various analyses (including constraints imposed by the optical map) provided a rough estimate of only ˜25 kbp on average of (peri)centromere per chromosome in C. zofingiensis.

The canonical rDNA repeat unit of C. zofingiensis became apparent early in assembly due to its presence in relatively high copy number. It assembled as a 9,702 bp circular contig annotated by RNAmmer 1.2 as ˜6.6 kbp 28S followed by ˜1.1 kbp of spacer followed by ˜1.8 kbp 18S followed by ˜0.2 kbp of spacer. From the presence of homologous sequence on chromosome 13 leading into the large sequencing gap of that chromosome, the optical tandem repeat that begins that sequencing gap, and the presence of two BamHI sites in the consensus rDNA unit (creating alternating fragments of ˜6.0 kbp and ˜3.7 kbp that are consistent with the optical tandem repeat), it is estimated that ˜24× tandem copies of the rDNA unit predominate in the first ˜40% of the large sequence gap of chromosome 13. Various analyses (e.g., Table 1) assume 24 exact copies begin this gap. The estimated number of copies is similar to M. neglectum, but much less than in the large genomes of A. thaliana and C. reinhardtii. The presence of other arrays of rDNA besides that of chromosome 13 cannot be completely ruled out (for example, neither sequence assembly nor the optical map reached one end of chromosome 5).

Genome Annotation and Transcriptomics

To facilitate annotation, we generated a C. zofingiensis transcriptome using RNA-Seq data collected from cells grown under 14 diverse conditions designed to capture a significant fraction of the cell's transcriptional repertoire (SI section). These conditions included treatments of different light intensities, nutrient limitations, and oxidative stress. Paired-end sequencing of transcriptome libraries was performed to facilitate determination of splice junctions, resolve close paralogous families, and de novo assembly (used as part of training the AUGUSTUS ab initio gene caller). In order to capture non-polyadenylated transcripts such as those from mitochondria and the chloroplast, libraries were prepared from total RNA depleted of rRNA.

RNA-Seq coverage, in conjunction with the de novo transcriptome assembly, was used to select a gene prediction method for producing gene models. Multiple pipelines, including Softberry's Fgenesh, MAKER (24), and AUGUSTUS (25) were evaluated using metrics such as RNA-Seq coverage capture and intron/exon boundary correlation with coverage. Of all evaluated pipelines, we selected AUGUSTUS as trained on the de novo transcriptome, which identified 15,274 nucleus-encoded protein-coding genes of which 15,194 are apparently complete.

When the RNA-Seq libraries were aligned to the genome assembly, 95±2% of reads aligned uniquely (mean±SD, N=10) and an additional 3±1% aligned to multiple locations, suggesting that the genome assembly represents nearly all coding genes. Further, 55±3% of RNA-Seq reads overlap by at least 80% with the coding portion of a gene model on the correct strand (mean±SD, N=10); only 1.3±0.3% overlap with a gene model on the opposite strand. Current gene models do not include 5′ and 3′ UTRs; extending gene models 1000 bp upstream and downstream increases the percentage of reads aligning to the correct strand to 96±2%.

To further quantify the completeness of the assembly and annotations, an analysis was performed by BUSCO (26) to identify C. zofingiensis orthologs for a set of 302 genes commonly found in eukaryotes. Orthologs were identified for 90.8% of these genes in the genome, with 98% of those genes judged to be complete by BUSCO (FIG. 3B, SI section). At the proteome level, orthologs were identified for 93.1% of the set, with 90.1% of those judged to be complete. BUSCO analyses on the other photosynthetic organisms show that C. zofingiensis gene model quality is comparable to that of C. subellipsoidea C-169 and C. sp. NC64A, superior to M. neglectum but inferior to model organisms C. reinhardtii and A. thaliana (FIG. 2B). Moreover, these analyses show that C. zofingiensis genome quality is higher than other algae and has less fragmented and missing orthologs (FIG. 2B). As expected, A. thaliana has many more repeated genes orthologs than the algae (FIG. 2B).

The sequences of all C. zofingiensis genes submitted to the NCBI nucleotide database were compared to the assembly presented here. For 12 out of 13 different genes, there was 99% or greater identity and 1% or fewer gaps as determined by BLAST alignment to the genome (SI section, Table S4). Only one, heat shock protein 70 (accession AY072815.1), had limited homology, but was isolated from a different strain.

C. zofingiensis contains the highest predicted fraction (˜39%) of protein-coding sequence of the six organisms in Table 1. The average length of its coding sequences (˜482 aa) is the longest apart from outlier C. reinhardtii, which helps bring C. reinhardtii to almost as high a fraction of coding sequence even though its genome is much larger. The median length of C. zofingiensis coding sequences (˜347 aa) is, however, more ordinary. The length of individual coding exons (whether by mean ˜291 bp or median ˜194 bp) of C. zofingiensis is the longest among the six organisms, while the mean (˜5.0) and median (4) number of coding exons per gene is low, being more similar to M. neglectum and A. thaliana rather than the higher numbers seen in the other algae. The number of identified tRNA loci (75 and forming a complete set for the standard amino acids) is moderate like C. subelhpsoidea C-169, rather than very low as for C. sp. NC64A and M. neglectum, or high for the two large genomes of C. reinhardtii and A. thaliana.

To compare the C. zofingiensis proteome to others in the green lineage, we functionally annotated gene models by forming families of genes across the six organisms of Table 1 using a method based on reciprocal near-best global amino acid alignments (SI section). This analysis generally permits one, many, or no genes per organism per family and separates genes into closer “primary” (putative orthologs) vs. further “additional” relationships (putative paralogs). The result contains 10,490 families involving more than one organism, of which 7,904 involve at most two genes per organism. There are some large families, with various histones constituting the largest families. ˜73% of C. zofingiensis genes (and ≥˜60% of every genome) are placed in a family involving multiple organisms. All six genomes (including C. zofingiensis) show evidence of tandem duplication of genes. A phylogram (FIG. 2A) estimated from putative 1:1:1:1:1:1 orthologs placed C. zofingiensis closest to M. neglectum and then C. reinhardtii (also in agreement with counts of C. zofingiensis genes as partitioned by their status as regards representation in M. neglectum, C. reinhardtii, and A. thaliana; FIG. 2B), forming a three-member clade that joins a two-member clade containing C. subelhpsoidea C-169 and C. sp. NC64A, consistent with existing literature (27), and this whole-genome data analysis is in agreement with placing this alga into the genus Chromochloris (7).

Although we do not find large stretches of nucleotide synteny between C. zofingiensis and the other genomes, we do find among all members in the green algal lineage (except for M. neglectum, whose current assembly is too fragmented for such an analysis) highly significant genomically localized blocks of genes in putative orthologous relationships (FIG. 2C and SI section), extending the result for C. subellipsoidea C-169 vs. C. sp. NC64A (FIG. 1). While block boundaries are rather well defined, gene order and coding strands within blocks are generally completely scrambled. It is likely that the blocks represent random chromosomal rearrangements that accumulate over time and diverge after speciation. Synteny analyses were also used to evaluate evolution of the green lineage, which resulted in topology consistent with the phylogram presented (FIG. 2, SI section).

To gain more insight into the metabolic function and cellular processes associated with specific proteins, we used in silico methods to predict subcellular localization of proteins encoded by the nuclear genome of C. zofingiensis. Using PredAlgo, an algal-specific subcellular localization prediction program trained on C. reinhardtii (28), we predicted nucleus-encoded proteins to distribute as ˜15% to the secretory system, ˜12% to the chloroplast, and ˜10% to mitochondria. The majority of proteins (˜63%) were predicted to be localized to other areas, which may be due to unidentified transit peptides, or the transit peptides of C. zofingiensis being significantly different from PredAlgo's C. reinhardtii training set. Additionally, errors in gene models, especially in terminal regions, may result in inaccurate localization predictions. The predicted distribution is similar to what has been noted for C. reinhardtii (29).

Mitochondrial and chloroplast genes were highly expressed over a wide range of conditions (SI section). Despite the organellar genomes being significantly smaller and expressing many fewer genes, the transcripts expressed by the chloroplast and mitochondria represent a substantial portion of the total cellular mRNA. In an analysis of the transcriptomic data from 14 diverse growth conditions, 31±9% and 7±2% of total RNA-Seq reads uniquely mapped to the chloroplast and mitochondrion genomes, respectively (mean±SD). With few genes, this translates to dramatically higher expression per gene: for the 73 protein-coding genes encoded in the chloroplast and 22 in the mitochondria, median transcript abundance across conditions was 686 and 419 FPKMs, respectively, in contrast to 5 FPKMs across all nuclear-encoded genes.

To identify genes that were more highly regulated under specific conditions, we compared expression of every gene over the 14 conditions and selected those with z-scores beyond ±2, plotting these as heatmaps (SI section, data not shown). The most prominent treatment to affect nuclear and plastid gene expression was oxidative stress by hydrogen peroxide (SI section), which significantly affected 3,934 genes. These genes were enriched for ABC-transporter domains (p=1.0×10⁻⁶), suggesting that export of toxics and xenobiotics is a significant mechanism for handling environmental stress in C. zofingiensis. Similarly, singlet oxygen stress induced by the chemical Rose Bengal affected 1,477 genes (SI section) and heterotrophic growth on glucose identified 853 genes (SI section). Nutrient deprivation had similar effects on most genes and far fewer genes were identified by analyses; for example, only 21 genes were detected as highly enriched in the iron-deficient sample.

Cryptic Sex and Motility in C. zofingiensis.

While C. zofingiensis has long been assumed to be asexual and non-motile, we investigated the presence of putative cilia/flagella and meiosis genes in its genome via the computationally identified gene families in conjunction with examination of associated gene expression across our conditions. The sequencing of the genome of C. sp. NC64A established a precedent for this type of analysis in green algae; similar to C. zofingiensis, no sexual cycle nor flagella has been observed in C. sp. NC64A, yet its genome revealed meiosis-specific and primarily motile flagella genes suggesting a cryptic sexual cycle (30). In the C. zofingiensis genome, we found putative orthologs of 73 of 78 genes (˜94%) in the CiliaCut (31) suggesting that it is likely that there could be a previously unobserved motile life cycle stage with flagella in this organism. C. zofingiensis was missing only five genes: DLC4, FAP111, FBB5, IFT20, and Tctex1 (all gene symbols in this work are with implicit “[v5.2]” suffixes). In C. reinhardtii, the ift20 deletion mutant lacks flagella and is immotile (32), but perhaps C. zofingiensis has an as-yet unidentified gene with similar function. C. zofingiensis does seem to have critical C. reinhardtii genes for flagella motility (FLA14, 33) and forming flagella (PF15, PF19), including conservation of functional residues in these two genes (34, 35). Additionally, FLA14, PF15, and PF19 were expressed in a variety of conditions, which suggests that these genes are functional despite lacking a visible flagella. Furthermore, we identified putative orthologs of 25 of 40 C. reinhardtii meiosis-associated genes (30, 36), which was more than we observed for C. sp. NC64A (only 22 of 40). In C. zofingiensis, most of these genes are transcribed under many conditions, but a few such as GSP1, MER3, and DMC1 had low transcript abundance except under a low dose of Rose Bengal (5 μM Rose Bengal, 0.5 h dark and 1 h 100 μmol photons m⁻²s⁻¹). Eleven of the families not found in C. zofingiensis were specific to C. reinhardtii. While these data cannot rule out the possibility of that a sexual cycle was recently lost, it is more likely that the high number of apparent cilia/flagella and meiosis genes suggest the existence of sexual reproduction and a motile stage that has not yet been observed in C. zofingiensis. Life cycle studies and in particular investigations for a cryptic sexual cycle, which may require specific conditions, should be the subject of future research in C. zofingiensis.

Astaxanthin Biosynthesis Pathway and Astaxanthin-Deficient Mutants.

Astaxanthin is an important and valuable algal bioproduct. In microalgae, astaxanthin is often produced in high abundance under stressful conditions, consistent with the hypothesis that it confers protection against oxidative stress. However, astaxanthin is not coupled functionally or structurally to the photosynthetic apparatus. Instead, astaxanthin functions as an internal sunscreen and antioxidant by absorbing excess light and quenching reactive oxygen species (13, 15). Additionally, astaxanthin accumulates in cytoplasmic lipid droplets where it could prevent peroxidation of fatty acids (13, 15). Astaxanthin is synthesized via the carotenoid biosynthetic pathway, which has been previously reviewed (15, 37, 38); however, key steps in its biosynthesis are still undetermined. Most of what is known about astaxanthin biosynthesis in algae comes from studies of H. pluvialis, for which we lack a sequenced genome. It is thought that β-carotene is exported from the chloroplast into lipid droplets in H. pluvialis where astaxanthin is synthesized by the introduction of two keto-groups catalyzed by a di-iron beta-ketolase (BKT), which is followed by the introduction of two hydroxyl groups catalyzed by a hydroxylase (CHYB) (15, 39). However, the mechanisms of export and transport remain elusive. In contrast, it is hypothesized that, in C. zofingiensis, the hydroxylation of β-carotene occurs first and that astaxanthin is formed by the ketolation of zeaxanthin (13). In vitro enzymatic studies of C. zofingiensis genes show that BKT catalyzes the ketolation of β-carotene to canthaxanthin and zeaxanthin to astaxanthin, while CHYB catalyzes the hydroxylation of β-carotene to zeaxanthin but not of canthaxanthin to astaxanthin (13). Liu et al. (13) also concluded there was only one copy of BKT and CHYB present in C. zofingiensis, however a recent study suggests there are two copies of BKT (40). For comparison, H. pluvialis has three BKT genes that are differentially regulated by environmental factors (41). In both microalgae, astaxanthin is esterified and stored in lipid droplets, however the acyltransferase enzyme involved has not been identified.

The genome of C. zofingiensis provides new insights into the astaxanthin pathway. The annotated carotenoid biosynthetic pathway in this alga (SI section, Table S2) appears to be very similar to that in C. reinhardtii (42). For example, there are four putative carotene hydroxylase genes, encoding three cytochrome P450s (two CYP97A and one CYP97C) and one di-iron type hydroxylase (CHYB). In addition, we found two putative BKT genes in the genome, in accordance with recent results (40). BKT1 and BKT2 contain highly conserved histidine motifs present in H. pluvialis and bacterial beta-ketolases (43, 44). These motifs are involved in iron binding and, in bacteria, mutations in them abolish the ability to form ketocarotenoids (44). The BKT genes from microalgae share highly conserved regions and are more similar to each other than those from bacteria. PredAlgo predicts the localization of both BKT1 and BKT2 to “other” areas of the cell, which could support localization of these enzymes to the cytosol. However, this prediction has not yet been verified experimentally. The genome also shows that there is a wide distribution of carotenoid biosynthesis genes across many chromosomes as is typical in eukaryotes.

To study C. zofingiensis astaxanthin production using a non-biased approach, a genetic screen was conducted to identify genes essential for astaxanthin synthesis. C. zofingiensis cells were randomly mutated using ultraviolet radiation, grown on glucose medium to induce astaxanthin accumulation (13), and 65 colonies were identified that were visibly green rather than pink due to lack of astaxanthin production, which was subsequently confirmed by HPLC analysis (SI section). Similar HPLC chromatograms were observed for all mutants (data not shown). Initially, three strains were selected for sequencing of BKT1 and BKT2, and all three showed different single mutations in highly conserved areas of BKT1 but no mutations in BKT2, and thus these mutants were named bkt1-1, bkt1-2, and bkt1-3 (SI Appendix, Table S3, Table S4). When grown in high light, the mutants accumulated increased levels of astaxanthin precursor compounds, especially zeaxanthin but also β-carotene, as well as more violaxanthin, despite similar levels of other pigments (FIG. 3, SI section). BKT1 was sequenced in an additional 13 mutants, and all showed mutations in conserved regions of the gene (SI section, Table S3). These data suggest that the disruption of BKT1 alone is sufficient to abolish astaxanthin production, but we cannot unambiguously distinguish if the committed step toward astaxanthin begins with β-carotene or zeaxanthin. While the screen demonstrates that the BKT1 enzyme is required for astaxanthin biosynthesis, based on these results we cannot determine if BKT2 is non-functional or if it may act in a secondary reaction downstream of BKT1. Both BKT1 and BKT2 were highly expressed in response to H₂O₂-stress (876 and 367 FPKMs, respectively), and both were identified in the screen for H₂O₂ treatment-enriched genes (SI Appendix, Fig S12). To a lesser extent, both were expressed in response to Rose Bengal treatment (394 and 35 FPKMs). It is unlikely that BKT1 and BKT2 form an obligate heterodimer or function in parallel pathways, as then mutations in either BKT1 or BKT2 should have been detected in different mutant strains.

High Light Induced Gene Expression.

To investigate the physiological changes associated with acclimation to high light and to elucidate unidentified genes in the astaxanthin biosynthesis pathway in C. zofingiensis, an RNA-Seq experiment was conducted in which cultures were moved from normal growth light intensity (100 μmol photons m⁻²s⁻¹) to high light intensity (400 μmol photons m⁻²s⁻¹) (SI Appendix, SI Text). Cultures were collected for nuclear, plastid, and mitochondrial gene expression analyses at 0, 0.5, 1, 3, 6, and 12 h (N=4) after the shift to high light, as well as in control cultures, which were maintained at the normal growth light intensity (SI section).

A principal component analysis of the regularized log₂-transformed counts from the resulting transcriptome profiles shows that time and treatment explain nearly all observed variation in gene expression between the conditions (95%, FIG. 4A). Time induces the largest variation for both the control and treatment cultures, which may have been caused by the diurnal lighting regime; these cultures were maintained on a day-night cycle (16 h light, 8 h dark) with sampling during daylight hours. The large changes throughout the day are not surprising given that in C. reinhardtii over 80% of the transcriptome is differentially expressed with diurnal periodicity (45). In addition to time-of-day changes, there is also a substantial effect from the shift to high light, as evidenced by the distinct groupings of control and treatment samples. Control cultures at each time point were used to separate the effects of time from high light.

To further evaluate the effect of high light, differentially expressed genes at each time point were identified. Those genes whose expression had a greater than two-fold change (p<0.01) in either direction between the high light-treated cultures and controls were determined and visualized in a heatmap, scaled relative to the number of genes in each group (FIG. 4B). Most genes were differentially expressed either early in the experiment (276 genes at 0.5 h and 492 genes at 1 h) or late (362 genes at 12 h). The greatest overlap of significantly differentially expressed genes was during the early time points (0.5 and 1 h), unsurprising given that these samples were collected closest together in time. Additionally, during these early time points, high light had a greater effect than time (FIG. 4A). Over the course of the experiment, there was greater upregulation of significantly differentially expressed genes with 67%, 75%, and 94% of genes upregulated at 0.5, 6, and 12 h, respectively, but more genes were significantly downregulated at 1 h (52%) and 3 h (86%). Most genes had relatively modest changes (<4 fold) in the cultures shifted to high light, although expression of ELIP8 (early light-induced protein) and ELIP10 had >20-fold increases at 0.5 h. Among chloroplast-encoded genes, both psaA and atpF had significant upregulation at 1 h. No significantly differentially expressed mitochondrial genes were found during the shift to high light.

Because of the high level of interest in astaxanthin production in C. zofingiensis, we examined the genes involved in carotenoid biosynthesis during the shift to high light. High light causes an accumulation of secondary carotenoids (in particular, astaxanthin) in C. zofingiensis (46-48). In the present study, both BKT1 and BKT2 have the highest increase in gene expression, which occurs immediately after the light shift at 0.5 h (FIG. 5). Despite the increase in BKT2 gene expression in high light, its role in carotenoid biosynthesis has not been established. Many genes at various points in the carotenoid biosynthesis pathway were upregulated early (0.5 h and 1 h) in response to the high light treatment, including phytoene synthase (PSY) (FIG. 5), which catalyzes the committed step in carotenoid biosynthesis. Previous studies have reported similar upregulation of PSY, PDS, BKT, and CHYB at longer time points in response to increases in light (46, 49, 50). However, our study also revealed a significant increase in expression of ZDS at 1 h and a significant decrease in LCYE shortly after the shift to high light. Further, downregulation of many genes in the carotenoid biosynthesis pathway was observed at later time points (6 and 12 h) in both the treatment and control cultures; this is likely an effect of the diurnal cycle. A higher expression of carotenoid biosynthesis genes would support an increase in secondary carotenoids, but does not exclude the possibility that post-translational modifications of carotenoid biosynthetic enzymes may also account for the accumulation of secondary carotenoids during high light.

The high-quality genome and transcriptome we generated in combination with the high light RNA-Seq experiment allowed us to identify candidates for additional genes involved in astaxanthin biosynthesis and accumulation. As mentioned above, little is known about the mechanism of translocation of the astaxanthin precursor(s) out of the chloroplast, the hydroxylation of the astaxanthin precursor, transport of astaxanthin into lipid droplets, or the esterification of astaxanthin. We identified putative genes involved astaxanthin biosynthesis through examining the significantly differentially expressed genes with high increases in gene expression during the shift to high light for genes with protein activity compatible with hypothetical mechanisms of astaxanthin biosynthesis. Genes that are upregulated early during high light that may be implicated in the astaxanthin pathway include four ABC transporters (Cz04g21110, Cz05g17060, Cz09g27180, and Cz08g16130), two cytochrome P450 proteins (Cz10g28330 and Cz11g14160), and an acyltransferase (Cz02g29020). The ABC transporters may form a complex that exports the astaxanthin precursor(s) from the chloroplast. The cytochrome P450 proteins could be involved in hydroxylation of astaxanthin precursors in the cytosol, and the acyltransferase could be involved in esterification of astaxanthin.

In addition to changes in carotenoid biosynthesis, we also investigated other algal high light responses, including photoprotective mechanisms and chlorophyll metabolism. In photosynthetic organisms, excess light must be safely dissipated to prevent oxidative damage. C. reinhardtii transiently expresses PSBS at the onset of high light and LHCSR proteins accumulate under high light, and this accumulation is correlated with non-photochemical quenching capacity (51, 52). While C. reinhardtii has multiple copies of both LHCSR and PSBS (51, 52), we found only single copies of LHCSR and PSBS in C. zofingiensis, despite having high non-photochemical quenching capacity (9). As expected, both LHCSR and PSBS were upregulated at the early time points during the shift to high light and, in particular, at 1 h, which is consistent with observations of C. reinhardtii during the dark-to-light transition (45). Similar to the carotenoid biosynthesis genes, under the diurnal cycle LHCSR and PSBS are downregulated by the end of the day (6 and 12 h) in both conditions (FIG. 5). Reduction in chlorophyll is another common physiological response of algae exposed to high light (53). Accordingly, during the shift to high light, many C. zofingiensis genes involved in chlorophyll synthesis were downregulated and chlorophyll degradation genes were upregulated (FIG. 5). The combination of these would lead to a reduction in chlorophyll content either during acclimation or as a stress response to high light.

Annotation of Metabolic Pathways and Photosynthesis-Related Gene

Genes encoding homologs of the primary metabolic pathway enzymes involved with carbon, carotenoids, chlorophyll, fatty acids, and lipids, as well as proteins involved in the composition, assembly, and regulation of the photosynthetic apparatus, were preliminarily identified using the BLAT sequence search tool (54) against one of our C. zofingiensis draft genomes (SI section, Table S2). Based on the quality of the alignments and comparison to well-characterized, closely related plant and algal query sequences, the targeted gene models in C. zofingiensis were submitted as queries in reciprocal BLAST searches against the NCBI RefSeq non-redundant protein database to confirm coverage, domain architecture, and similarity across closely related homologs. Because of the high quality of the C. zofingiensis assembly, this procedure resulted in a nearly complete list of putative genes needed to complete each pathway. Identified gene models were used to assess the quality of the automated gene family analysis across the six species of Table 1, and the automated analysis was used to confirm additional candidate models and expand the set of annotations. Based on high sequence similarities and conservation of functional domains, we are generally confident in the assignments of homology for these models. However, it is possible that additional functional isoforms composed of more divergent sequences may also be present, having been missed by the parameters used for BLAT, BLAST, and the automated gene family analysis.

Identification and annotation of genes involved in lipid biosynthesis can provide targets for exploitation of C. zofingiensis for biofuel production. Using other oleaginous organisms as a guide, we would expect a robust oil-producing microalga to have an expanded family of acyltransferases. The C. zofingiensis diacylglycerol acyltransferases (DGAT) are too divergent from the protein sequences of Type 1 DGAT and DGTT (Type 2 DGATs, 55) in C. reinhardtii, A. thaliana, and M. neglectum to identify the corresponding genes via BLAT. Using a more sensitive BLAST search with both types of DGAT sequences from C. subelhpsoidea C-169 (gi|545360296), Chlorella vulgaris (gb|ALP13863.1), Nannochloropsis gaditana (gb|EWM23187.1), A. thaliana (gi|15224779, gi|18409359), and C. reinhardtii (Cre01.g045903, Cre03.g205050), additional copies of DGAT Type 1- and DGTT-encoding genes were identified in C. zofingiensis, and yet more were identified using the automated gene family analysis. In total, 11 genes were identified that have either an LPLAT (lysophospholipid acyltransferase) domain or a closely related MBOAT (membrane bound O-acyltransferase) domain (SI Appendix, Table S2). We have tentatively assigned these genes as encoding proteins with diacylglycerol acyltransferase activity, however some of these C. zofingiensis gene models have higher similarity to predicted proteins of unknown function than to annotated Type 1 or Type 2 DGAT proteins from other closely related organisms. Our finding of multiple copies of putative DGAT and DGTT genes in C. zofingiensis is consistent with transcriptome results from the closely related ATCC 30412 strain (40). Of course, it is also possible, though unlikely, that one or multiple additional copies of DGAT or DGTT may be yet unidentified due to a gene modeling or assembly problem.

Homologous gene models were also identified for components of the photosynthetic apparatus and its assembly, including proteins that compose PSI, PSII, the major and minor light harvesting antennae, the cytochrome b₆f complex, the chloroplast ATP synthase complex, and soluble electron carriers, as well as known assembly factors for these complexes (SI Appendix, Table S2). Thirteen of the C. zofingiensis light-harvesting complex (LHC) genes are predicted to be more like PSI-associated LHC genes (LHCAs) rather than nine PSII-associated LHC genes (LHCBs) (SI Appendix, Table S2), in contrast to the distribution found in C. reinhardtii, which has nine of each of LHCA and LHCB. Further experimental work is needed to confirm the expression profiles and photosystem association of each of these putative LHC proteins, especially under different light and stress conditions. Of note is one LHC model (Lhcb-like3, Cz04g24050) with little to no transcriptional expression detected in any of our RNA-Seq conditions.

Brief Summary of Above Examples

Our analyses of the C. zofingiensis genome, transcriptome, astaxanthin-deficient mutants, and RNA expression changes under high light reveal new insights into the basic biology of the green lineage of photosynthetic organisms and the carotenoid biosynthesis pathway. We present a high-quality chromosome-level assembly with independent genome validation including identification of (peri)centromeric loci for each chromosome and an assembly extending into telomere-associated tips for the majority of chromosomes. The compact ˜58 Mbp genome has balanced G+C content and is rich in protein-coding sequence with few long exons per gene and relatively little repetitive sequence. We identified ortholog families for the majority of C. zofingiensis genes. The gene density is uniform over chromosomes and a syntenic comparison with other algae uncovered highly significant genomically localized blocks of genes in putative orthologous relationships; however, gene order and strands within blocks are scrambled. We have shown that BKT1 is critical for the production of astaxanthin and have identified candidate genes that could be involved in missing steps in astaxanthin biosynthesis and accumulation. The addition of genomics to the experimental toolkit for C. zofingiensis makes it a very attractive alga not only for fundamental studies of its biology but also the economically viable and environmentally sustainable production of biofuels and important bioproducts.

Table 1 shows the features of the Chromochloris zofingiensis genome in comparison to selected previously sequenced genomes. The C. zofingiensis genome was compared to four other green algal genomes (Chlamydomonas reinhardtii, Coccomyxa subelhpsoidea C-169, Chlorella sp. NC64A, and Monoraphidium neglectum) and the model plant Arabidopsis thaliana. Quantities were generally computed with uniform rules applied to most recently available genome assemblies and annotation releases (SI Appendix, SI Text).

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Supplemental Information for Examples

Chromochloris zofingiensis Strains and Culture Conditions

Chromochloris zofingiensis strain SAG 211-14 was obtained from the Culture Collection of Algae at Goettingen University. The cells were grown at 25° C. in liquid cultures shaking at 100-150 rpm in diurnal (16 h light, 8 h dark) conditions with light intensity of 90-100 μmol photons m⁻²s⁻¹ unless stated otherwise. Cells were grown in Proteose Medium (UTEX Culture Collection of Algae) with Chu's micronutrient solution (2 mL/L, UTEX Culture Collection of Algae) unless stated otherwise. Cells were counted with the Multisizer 3 Coulter Counter (Beckman Coulter). Cells were harvested by centrifugation (2,200-4,620 g for 5-10 min), discarding the supernatant, resuspending the cells in media and transferring to an eppendorf tube, pelleting by centrifugation (15,000 g for 5 min), discarding the supernatant, and freezing the cell pellet in liquid nitrogen unless stated otherwise.

X-Ray Tomography

Cells were grown until log phase, pelleted by centrifugation (700 g for 2 min), and then loaded into custom-made thin-walled glass capillaries (1). Glass capillaries had been previously dipped in a solution of 100 nm gold nanoparticles (EMGC100, BBI International, Cardiff, CF14 5DX, UK), which were subsequently used as fiducial markers for alignment of the X-ray projections. Once loaded into capillaries, cells were cryo-preserved by plunging the tip of the specimen capillary into a ˜90 K reservoir of liquid propane at 2 m s⁻¹ using a custom-made fast-freezing apparatus.

Soft X-ray tomographic data were acquired using the cryogenic soft X-ray microscope in the National Center for X-ray Tomography (NCXT) at the Advanced Light Source in Berkeley, Calif. The microscope and image acquisition have been described in detail previously (2, 3). Projection images were collected at 517 eV using a Fresnel zone plate with a resolution of ˜50 nm as the objective lens. For each data set, 90 projection images were acquired spanning a range of 180°. During data acquisition, the specimen was kept in a stream of helium gas that had been cooled to liquid nitrogen temperatures to maintain cryo-preservation of the sample. Depending on the thickness of the specimen, exposure times for each projection image varied between 200 and 350 ms. 3-D reconstructions of the X-ray projections were calculated using the software package IMOD after manually tracking fiducial markers on adjacent images for alignment (4). AMIRA (FEI) was used to semi-automatically segment the 3-D volumetric reconstructions into subcellular compartments (lipid droplets, chloroplasts, starch, mitochondria) based on their different gray level ranges. Segmentation of the nucleus was performed manually.

DNA Preparation and Quality Assessment

Genomic DNA was prepared as follows. Total cellular DNA was extracted from cells grown in 1 L cultures to ˜5×10⁶ cells/mL. Harvested cells were resuspended in 300 μL Milli-Q purified water and 500 μL lysis buffer (100 mM Tris-HCl pH 8.0, 40 mM EDTA, 400 mM NaCl, 2% SDS) and incubated for 2 h at 65° C. while rotating. 170 μL of 5 M NaCl and 135 μL of 10% w/v CTAB in 700 mM NaCl were added. After incubation for 10 min, the DNA was extracted by adding phenol:chloroform, vigorously shaking, and centrifuging (˜15,000 g for 5 min) to separate phases. The aqueous phase was removed and placed in a new tube with 5 μL of RNase A, incubated for 20 min at 37° C., and followed by two additional phenol:chloroform extractions and one chloroform extraction. To precipitate the DNA, 0.1× sample volume of 5 M NaCl and 0.7× sample volume of isopropanol were added to the resulting aqueous phase, the sample was mixed, and pelleted by centrifugation (15,000 g for 15 min at 4° C.). The supernatant was removed and pellet was washed with cold 70% ethanol, centrifuged (15,000 g for 5 min at 4° C.), and the supernatant removed. The DNA was cleaned with an ethanol precipitation step (100% ethanol, 100 mM sodium acetate pH 5, overnight at 20° C.), centrifuged (˜15,000 g for 5 min at 4° C.), and followed by an ethanol wash. The DNA pellet was briefly air-dried and resupsended in Milli-Q purified water. DNA concentration and quality was assessed by optical absorbance on a NanoDrop 2000 spectrophotometer (Thermo Scientific).

To obtain high molecular weight DNA (≈270 Kbp) for optical mapping, 1 L of cells were grown to ˜5×10⁶ cells/ml. The harvested cell pellet was washed twice with cold ethanol and resuspended in buffer (200 mM NaCl, 100 mM EDTA, 10 mM Tris, pH 7.2). An equal volume of 1% agarose was gently mixed with the cells and the cell-agarose suspension was aliquoted into plug molds and cooled (4° C. for ˜60 min). The cell wall was digested by incubating the cell plugs in protoplasting solution (4% w/v hemicellulose, 2% w/v driselase, 1 M sorbitol, 5 mM sodium citrate, 240 mM EDTA pH 8.0, 10 mM 2-mercaptoethanol) overnight at 37° C. while shaking. To lyse the cells, the protoplasting solution was removed and the cell plugs were incubated in lysis solution (0.5 M EDTA pH 9.5, 1% w/v N-lauroylsarcosine, 5 mg/ml proteinase K) overnight at 50° C. The lysis solution was removed and the cell plugs placed in 0.5 M EDTA pH 9.5 and shipped to OpGen, Inc. for optical mapping using BamHI enzyme.

RNA Preparation and Quality Assessment

RNA was prepared as follows. Cells were washed with cold ethanol on dry ice and ethanol was removed by centrifugation (2,200 g for 3 min at 4° C.). To break cells open, cells were homogenized with lysing matrix D on dry ice for 2×60 s with the FastPrep-24 (6.0 m MP Biomedicals). Buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM EDTA, 2% SDS, 1 mg/mL proteinase K) was added, samples were vortexed and incubated for 3 min at room temperature, and cell debris was pelleted by centrifugation (20,000 g for 3 min). One mL of sample was added to 10 mL of TRIzol in MaXtract HD tube and incubated for 3 min at room temperature. To extract RNA, ⅕ volume chloroform was added, samples were vigorously shaken, incubated for 5 min at room temperature, and phases were separated by centrifugation (800 g for 5 min at 22° C.) and decanting. Total RNA was precipitated by adding cold ethanol on the aqueous phase and purified using the miRNeasy mini kit (Qiagen). RNA was eluted with DEPC-treated water and cleaned with an ethanol precipitation step (100% ethanol, 85 mM sodium acetate pH 8.0), centrifugation (˜15,000 g for 5 min at 4° C.), and ethanol washing. The pellet was briefly air-dried and resuspended in DEPC-treated water. RNA concentration and integrity was assessed by NanoDrop 2000 spectrophotometer (Thermo Scientific) and Agilent 2100 Bioanalyzer.

RNA-Seq

Total RNA was purified from each culture as described above. The rRNA was selectively depleted with the Ribo-Zero rRNA Removal Kit (Plant Leaf) according to the manufacturer's instructions (Illumina). The remaining RNA was converted into cDNA and made into sequence-ready libraries with the KAPA Stranded RNA-Seq Kit (Kapa Biosystems). The 14 de novo transcriptome RNA-Seq libraries were pooled and sequenced with 150+150 bp paired-end reads on two lanes of a HiSeq 2500 high-throughput sequencer according to manufacturer's instructions (Illumina). The 44 high light RNA-Seq libraries were combined into three pools and sequenced with 50 bp single-end reads on three lanes of a HiSeq 2500.

The resulting data was demultiplexed with in-house scripts. Adapter sequences were trimmed with Scythe (5) and aligned to the ChrZofV5 release of the C. zofingiensis genome with RNA STAR (6). Determination of counts per gene and transcript abundance in terms of fragments per Kbp of gene per million mapped fragments (FPKMs) were made with Cuffdiff (7). Further analyses and figures were generated with cummeRbund package in the R statistical computing environment (8). PCA was performed with plotPCA( ) from the R affy package (9). Two-fold differentially-expressed genes and regularized log₂-transformation were performed with the R DESeq2 package (10).

De Novo Transcriptome Conditions

Transcriptome material was derived from 100 mL cultures of cells (˜4-9×10⁶ cells/mL) from 14 different conditions: high light (400 μmol photons m⁻²s⁻¹), medium light (100 μmol photons m⁻²s⁻¹), low light (10 μmol photons m⁻²s⁻¹), glucose (20 mM), 48 h darkness, 4 h anaerobic, 4 h dark and anaerobic, 1 h without sulfur (Bristol's Medium without MgSO₄, UTEX Culture Collection of Algae), 1 h without nitrogen (Bristol's Medium without NaNO₃), 1 h without phosphorus (Bristol's Medium without K₂HPO₄, KH₂PO₄), 1 h without iron (Bristol's Medium), low oxidative stress (5 μM rose bengal, 0.5 h dark followed by 1 h 100 μmol photons m⁻²s⁻¹), high oxidative stress (5 μM rose bengal, 0.5 h dark followed by h 100 μmol photons m⁻²s⁻¹), and hydrogen peroxide oxidative stress (1 mM H₂O₂). Cells were collected by centrifugation (2,200 g for 5 min at 4° C.), the supernatant was discarded and the cell pellet was frozen in liquid nitrogen.

Changes in Gene Expression During Shift to High Light

The gene expression light intensity experiment from medium light (100 μmol photons m⁻²s⁻¹) to high light (400 μmol photons m⁻²s⁻¹) was conducted as follows. 1 L cell cultures were grown to log phase (˜3.0×10⁶ cells/mL) under medium light (100 μmol photons m⁻² s⁻¹). Cultures were mixed and divided into 75 mL cultures in sterile 250 mL beakers. After acclimating overnight, the light treatment cultures were moved from 100 μmol photons m⁻²s⁻¹ to 400 μmol photons m⁻²s⁻¹, while control cultures were maintained under 100 μmol photons m⁻²s⁻¹. Replicates (N=4) were collected at 0, 0.5, 1, 3, 6, and 12 h, harvested by centrifugation (200 g for 5 min at 4° C.), and frozen in liquid nitrogen. RNA was extracted, processed, and analyzed as described above.

Assembly Overview

Next-generation sequencing and associated software has made draft assemblies via short-read whole genome shotgun sequencing easy and relatively automatic. For eukaryotic organisms, these drafts are typically highly fragmentary by traditional standards of model organisms, with fragments often of size spanning only one to a few genes at a time. For Chromochloris, a chromosome-level assembly comparable to model organisms was aimed for, and initial drafts purely via automated short-read methods were only of “gene-space” quality and did not meet the goal. Hence, additional data—a global optical restriction fragment map from OpGen, Inc. and long reads via Pacific Biosystems (“PacBio”)—were collected. No software was found able to automatically incorporate this additional data well enough to meet the assembly goal; hence, extensive manual integration effort was expended to meet the goal starting from automated assemblies as a base.

Genomic and RNA-Seq Sequences

Two Illumina paired-end libraries—“S” with shorter and “L” with longer inserts were prepared as described earlier for genomic (combined nuclear, chloroplast, and mitochondrion) sequencing, including Illumina inline controls and a small amount of Illumina PhiX. Each library was run as an entire single lane of a HiSeq 2000 V3 flowcell at the UCLA BSCRC Sequencing Core to obtain ˜104M (“S”) and ˜66M (“L”) paired end 100+100 nt reads with ˜96% of pairs passing RTA PF=1 (PF=0 pairs were discarded). (Pacific Biosciences genomic reads are discussed later.)

Fourteen Illumina TruSeq paired-end RNA-Seq sub-libraries were prepared as described above. A single equi-molar pool was run on both lanes of a HiSeq 2500 V1 rapid flowcell at the UCLA BSCRC Sequencing Core to obtain ˜476M 151+151 nt read pairs with 7 nt TruSeq index reads with ˜86% of pairs passing RTA PF=1 (PF=0 pairs were discarded). Demultiplexing for assembly by perfect match to expected 7-mers gave ˜23M to ˜34M read pairs per sub-library and ˜397M (˜97% of PF=1) read pairs total.

Analyses of reads, a multitude of in silico-targeted subsets of reads, and various fractions of reads (e.g., heads or tails of first or second ends) were made over many iterations, starting with exploratory preliminary analyses under minimal assumptions and proceeding toward final analyses as conclusions and partial results accumulated. Tools used included assemblers Ray (11), ABySS (12), and ALLPATHS-LG (13, 14); aligners Bowtie (15), Bowtie2 (16), HISAT/HISAT2 (17), BLAST (18), BLAST+(19), LAST (20), LASTZ (21), BLAT (22), OpGen, Inc.'s MapSolver, BLASR (23), and Parasail (24); error correctors/double-sequenced end overlappers/adapter trimmers Proovread (25), SeqPrep (26), and Cutadapt (27); sequence analyzers Jellyfish (28), MUMmer (29), TRF (30), IRF (31), RepeatMasker (32) with Repbase Update (33), and RepeatModeler (34); gene callers AUGUSTUS (35) and tRNAscan-SE (36); visualization/analysis tools Savant (37), IGB (38), IGV (39), Biomatters Limited's Geneious, and Circos (40); GUI automaton Keyboard Maestro of Stairways Software Pty Ltd.; standard UNIX text-processing tools as well as bioinformatic utilities such as SAMtools (41), DEXTRACTOR (42), HTSeq (43), and EMBOSS (44); databases of biological knowledge such as NCBI (45), Pfam (46), and Rfam (47); as well as custom one-off programs and scripts written in languages such as C++, Perl, Wolfram's Mathematica, and MathWorks's MATLAB. Some computations were carried out on the UCLA Hoffman2 computing cluster.

Insert Lengths, and Read Preparation/Composition

From preliminary and later assemblies, mode insert lengths exclusive of adapters were ≈156 nt for “S” and ≈370 nt for “L”, with “S” fairly Gaussian with standard deviation ≈16 nt, but “L” bimodal with approximately one third in a wide mode at ≈200 nt and two thirds in a non-Gaussian narrower mode at ≈370 nt skewed longer. Inserts below 100 nt read into adapters (empirically verified to be as expected: for “S”, first end A+TruSeq #6+dark/poly-A, second end reverse complement of TruSeq universal adapter+dark/poly-A; for “L”, same except with TruSeq #12). Little of the “S” and “L” distributions is so short, and only ˜150K (<˜0.2%) of “S” and ˜271K (<˜0.5%) of “L” pairs contain ≥1 16-mer of consensus adapter ignoring dark/poly-A tails. Preliminary analyses often did not try to identify and remove adapters, while later analyses generally had them stripped via SeqPrep or Cutadapt.

Inserts below 200 nt have overlapping ends: almost all of the “S” distribution is as such, and a fraction of the shorter “L” mode is as well. Early analyses identified overlapped ends (merging double-sequencing to form consensus virtual single end reads) via unique overlaps of ≥16 nt with ≤3 mismatches; ˜88% of “S” pairs and ˜7% of “L” pairs were merged. The resulting pool of reads used for initial assemblies was then ˜89M and ˜5M virtual single end reads of total sizes ˜14 Gnt and ˜0.7 Gnt, and ˜12M and ˜59M read pairs of total sizes ˜2 Gnt and ˜12 Gnt, for a grand total of ˜28.6 Gnt. Later analyses used SeqPrep for overlap detection and merging.

Rough composition is ≈1.3%/1.0% of “S”/“L” pairs as Illumina inline process controls (with ˜95%/97% of pairs with ≥20% of 16-mers hitting a known control having ≥80% of 16-mers being hits) and ≈1.8%/2.9% as PhiX (with ˜94%98% of pairs with ≥5% of 16-mers hitting de novo circular PhiX having >⅔ of 16-mers being hits), leaving ≈97%/96% for nuclear genome+chloroplast+mitochondrion. Once organelle genomes became available, ≈0.5%/0.7% and ≈0.2%/0.2% was estimated as chloroplast and mitochondrion, respectively.

With (1) coverage plentiful relative to the ≈58 Mbp assembly size estimate (see next section), (2) ≈70%/80% of “S”/“L” PhiX read pairs manifestly error-free, and (3) several dozen not unlikely corruption possibilities of comparable probability existing for a typical read (e.g., although PhiX errors concentrated as expected at the tails of reads, error position probability was substantial across more than 20 nt), it was decided to not generally perform spectral-based read “error correction” procedures on the Illumina reads. (However, correction of the Pacific Biosciences reads was critical for their use in refining the assembly.)

Nuclear Assembly Phases 1 and 2: Automated Base Assemblies

Histograms of the number of times distinct strand-collapsed (e.g., Jellyfish “canonical”) k-mers appeared in the prepared Illumina read pool for various k suggested that potential diploidy was not a great concern, multi-copy repeats (although surely present) did not constitute an excessive fraction of the genome, and there were no large contaminants (e.g., bacterial genomes), suggestions later supported by data such as the ˜58 Mbp optical size estimate and BLAST comparisons of final genome products against the universe of NCBI sequences. Plateaus visible in the cumulative plot provided one of the filterable signals by which the assembly of non-chromosomal sequences began.

The main automated draft assembly used in the first years of the project (“Phase 1”) was an ABySS k=95 “gene-space” one on the prepared Illumina reads consisting of 3,513 scaffolds with longest ˜407 Kbp, N50 ˜79 Kbp, N90 ˜19 Kbp, L50=217, and L90=754. This assembly guided further decisions (e.g., use of optical mapping and with BamHI) and was the point at which downstream analyses such as gene prediction began. Because Phase 1 contigs were slightly shorter than needed for high-likelihood automatic optical map placement, in Phase 2 additional assemblers were tried in an effort to find a slightly better automated base; a Ray k=51 “gene-space” one on the prepared Illumina reads consisting of 1,335 contigs with longest ˜479 Kbp, N50 ˜88 Kbp, N90 ˜25 Kbp, L50=193, and L90=652 was chosen.

Nuclear Assembly Phase 3: Optical Map and Chromosome-Level Scaffolding, Joining, Filling

OpGen, Inc. was contracted to construct an “optical map” of Chromochloris by imaging immobilized complete restriction digests of linearly-combed large molecular weight pieces (“hunks”) of genomic DNA we provided. Based on Phase 1, they chose BamHI (G|GATCC) as digest enzyme due to the range of predicted fragment lengths being mostly accessible to their technology. They ran 12 high-density MapCards to obtain approximate fragment length fingerprints for ˜318K hunks, which they assembled into 19 maptig chromosomes of total size ˜58 Mbp whose constitutents are not A/C/G/T nucleotide calls, but approximate fragment lengths under complete BamHI digestion (SI Files S6-S7). (As they omit all small maptigs, chloroplast and mitochondrion do not appear.) The final nuclear genome nucleotide sequences described in this work—the “ChrZofV5” version 5 release that Phase 4 (described later) ends with—adopt chromosome numbering and ‘+’ strand decisions from this optical assembly.

During optical assembly, hunk fingerprints are piled up in multiple alignments with typical coverage of several dozens; chromosome ends are manifest as consensus locations beyond which hunk fingerprints do not extend (up to uncertainty in optically-estimated fragment lengths). OpGen observed both ends of all chromosomes except the right end of chromosome 5, the tail of which assembled into an approximate optical inverted repeat that, as discussed further later, likely is just the beginning of a much longer true sequence inverted repeat. (Due to this, the optical length of chromosome 5 is likely underestimated by ˜0.56 Mbp and chromosome numbering does not reflect true size largest to smallest.)

OpGen's MapSolver software visualizes the optical map and aligns sequence contigs/scaffolds to it. (A degree of mismatch is allowed due to optical length uncertainty, the tendency of small fragments to be lost optically, and the possibility of small basecall sequence errors creating or deleting cutsites.) Experience suggests a contig/scaffold needs ≥5 interior fragments of non-small length (>Kbp) for MapSolver to have a reasonable probability of placing it. This translates into a wide variety of contig/scaffold lengths due to Chromochloris BamHI fragment size variation, and Phase 1 scaffolds were often near this threshold with only ˜12% covering ˜37.6 Mbp being automatically placeable, even allowing non-unique placements and multiple coverage; the slightly longer contigs of Phase 2 improved to ˜29% covering ˜38.2 Mbp. However, by Phase 4's end with extensive hand work, ˜93% of the optical map was uniquely covered (see main text FIG. 1) with just a single sequence scaffold per optical chromosome.

Most automated assemblers have as a design goal to be conservative, in that they would prefer to give a more fragmented result (which could be pasted together in an unknown way to get “truth”) rather than one with mis-assemblies (in which some contigs/scaffolds would need to be taken apart before pasting could arrive at truth). Consistent with this, only a handful of Phase 1/2 scaffolds/contigs were found to be mis-assemblies via alignment to the optical map, increasing confidence that base sequence at finer resolution than the optical map was generally correct.

The per-chromosome single sequence scaffolds were formed from iterative rounds of optical placements of smaller subsequences (longer and longer as hand work proceeded), with the optical map providing global, externally-validated subsequence ordering and strand orientations and enabling approximate but accurately-sized N-filled gaps among subsequences and chromosome edges. Placements that resulted in overlapping or touching subsequences up to optical length uncertainty were, e.g., inspected at the sequence level for nucleotide overlap agreement of shorter lengths than automatic assemblers might otherwise require; reads and read pairs (including Pacific Biosystems long reads once Phase 4 began) touching and spanning gaps were isolated; and ambiguous placements could sometimes be resolved in favor of those not covering already well-covered parts of the optical map.

The optical map was useful: as hand work proceeded, speculative contig joins and extensions became reliable, as once further BamHI sites were reached, independent verification by the optical map was attained and possibilities were eliminated. Similar to the physical maps used in model organism projects, the optical map provided a global ground truth and acted as a ratchet for making positive progress that kept hand work from compounding mistakes. Each additional placement generally made other placements easier, as one could focus on gaps and not only were gaps getting fewer and smaller, but the pile of contigs, scaffolds, and reads to fill them with was also shrinking. Sources for speculation included: alignments of contigs and scaffolds to themselves; re-alignments of reads and read pairs to contigs and scaffolds; and, most importantly, the Pacific Biosciences long reads of Phase 4. Consensuses of supporting evidence spanning gaps was used to fill gaps; in some cases, these gap fills are of low quality (e.g., naked single-read PacBio sequence) but as long as the evidence supporting the join was substantial it was preferable to provide some representative sequence and close gaps rather than fret for first public release over every basepair being absolutely certain.

Nuclear Assembly Phase 4: Pacific Biosciences Reads, Contig Joining, and Gap Filling

Numerous barriers in the draft assemblies evidently arose from the use of only short, paired end Illumina reads. Many difficulties were near repetitive sequence, either: (1) non-short segments of moderate/high entropy DNA that occur multiple times in the genome; or (2) low entropy DNA (e.g., microsatellites), these being trouble because of either (a) ambiguous continuations due to only having short reads, or (b) coverage collapse of multiple orders of magnitude (even so far as to completely deplete our nearly half-a-thousand-fold average coverage). Difficulties of type (2b) were common at points of very high G+C content as short as a dozen or two basepairs (as has been the authors' experience on other projects with the Illumina platform), and there appear to be many such loci in this genome (ChrZofV5 has 191 clusters of G+C runs of length ≥24 nt).

To overcome some of these obstacles and better scaffold subsequences, four 75 fps 3 h PacBio RS-II/Springfield 1.1 runs of genomic DNA with BluePippin selection were performed at the DNA Sequencing & Genotyping Center of the Delaware Biotechnology Institute to obtain long reads, but of relatively low quality. Each SMRT cell contained 163,482 ZMWs (“wells”). Of wells with ≥1 insert called by the PacBio basecaller, ˜94% had only a single insert; hence, only a single longest interval per well was retained from the intersection of the “insert” and “HQ” regions, and no circular consensuses were made. The result was 149,364 “subreads” (from ˜30% of wells) of 12 nt to ˜34 Knt (median ˜3.1 Knt) of total length ˜692 Mbp.

Per-base error rates were estimated by the PacBio basecaller as very high compared to Illumina reads and as mostly indels rather than substitutions. No subread basecalls were given combined substitute/insert/delete/merge Phred qualities ≥15 (˜3% chance of error or better), the mode was Phred 13 (˜5% error), ˜25% had Phred quality 0 to 7 (˜20% to ˜100% error), and the average chance of error per base was ˜18%. Hence, pre-correction alignments of subreads to assemblies used PacBio-aware BLASR. While each default 12-mer seed only has ≈9% chance of being uncorrupted, queries ≥≈220 nt long have estimated chance ≥≈99% of ≥1 uncorrupted seed. Most BLASR parameters were left at defaults, but filtering was lowered to impose no minimum read/subread length and no percent identity requirement, and best alignments per query was raised to 250 internal and 100 emit as (1) the shortest target contigs were ≈200 bp and longest PacBio reads ≈50× longer; and (2) alignments descending into false positives were desired so that their statistics could be inferred from their great numbers.

From histograms of query and target alignment spans, a threshold of ≥140 nt was chosen for both spans to separate most true hits from very short false positives as well as short sequences repetitively occuring in Chromochloris, resulting in alignments. (Repetitive sequences of longer lengths remained; pre-alignment masking by tandem repeat finder TRF was sometimes used to help.) In subreads with ≥1 alignment, ˜88%/˜7%/˜5% of PacBio bases on average participated in exactly one/zero/multiple alignments. For draft contigs participating in ≥1 alignment, mode coverage by PacBio bases was typically 8, with ≈0.1%/≈0.6%/≈97% of bases uncovered/covered exactly once/covered 2 to 23 times. PacBio reads did not show nearly as much coverage variation across sequence the Illumina platform found difficult (e.g., at runs of G+C's). The top alignment by BLASR score per subread was enriched for near-full length alignment span on the query. Once organelle genomes became available (discussed later), estimates put ≈0.1%/≈0.2% of aligning subreads as mitochondrion/chloroplast.

PacBio subread alignments were repeatedly used to help make assembly subsequence joins and to fill gaps as mentioned in Phase 3. A typical pass began by extrapolation of the unaligned ends of each aligned subread by the average compression/expansion ratio from indels in the aligned portions. Extrapolations might (“overhang”) or might not extend beyond a subsequence's boundary, but overhangs of ≥1 Knt were not uncommon and, similar to earlier filtering, those of ≥140 nt were deemed “interesting”. Based on histograms of distance of alignment starts and stops to subsequence edges for subreads with interesting overhangs, it was decided to consider alignment starts and stops within 9 bp of a subsequence's edge as having reached the edge. A subread alignment with an interesting overhang to a subsequence was considered “linkable” if it reached the same end of the subsequence (both ends for those with interesting overhangs on both ends). Subreads with a single linkable alignment on each end and to different subsequences on each end were declared “linking”; each of these suggests a merging of two subsequences with a particular relative distance and orientation with explicit sequence to fill any gap. Suggested merges from linking subreads were collected into a directed graph (that was typically enriched for linear paths) and evidence weighed at nodes with multiple incident arcs to determine if one arc had much more support (e.g., 6-fold more) than others, in which case only the dominant arc was retained and otherwise all arcs removed. The resulting directed graph of linear chains provides a round of up to a few hundred tentative assembly subsequence joins and gap fills to participate in the hand work process discussed in Phase 3. It was always satisfying to merge two or several subsequences into a subsequence large enough that optical placement became probable, and then finding the new subsequence had a unique optical placement that perfectly filled a hole in existing placements.

Using the pool of prepared but unassembled Illumina short reads as reference, the Proovread error corrector was also run on PacBio reads from three of the SMRT cells to obtain 83,069 polished trimmed reads of total length ˜292 Mnt whose lengths were primarily between 500 nt and ˜21 Knt (median ˜3.0 Knt), with almost all bases explicit A/C/G/Ts (rare isolated Ns) and almost all per-base Phred-scale quality scores ≥19 (≈1 in 79 chance of error or better). These were very useful, as they enabled use of non-PacBio-aware tools (BLAST) to query and manipulate the long read dataset, and were used both in ways similar to the uncorrected reads (e.g., in procedures like the previous paragraph) as well as more targeted questions that arose once two subsequences were placed near each other on the optical map. (During operations such as subsequence joining, the larger pile of untrimmed corrected reads also produced by Proovread was queried as well; in certain cases, this was the only way to make progress and gap fill exposes naked single-read PacBio sequence.)

Periodically, and one last time at the end of Phase 4, prepared Illumina reads not aligning to the working assembly were re-de novo assembled to maintain an accurate pool of unplaced contigs/scaffolds. Only those of length ≥1 Kbp with less than one third of their 31-mers already represented were retained for the final chrUn##### unplaced contigs/scaffolds in the ChrZofV5 assembly release. To simplify naming, a few had a small number of Ns suffixed to make all their lengths unique.

Overall Structure of the Nuclear Genome

Telomeres.

As Phases 3 and 4 progressed and chromosome-level contigs/scaffolds approached optical ends of a chromosome, junctions with telomere repeats became apparent, and efforts were made (returning to Illumina and PacBio reads as necessary) to extend all sequences near such junctions at least partially beyond the junctions. As evident from chromosomes 1-4, 6-9, 13, 15, and 18-19 of the final ChrZofV5 assembly, the canonical Chromochloris telomeric repeat is apparently (CCCTAAA)_(n) at 5′-ends of chromosome strands, and from chromosomes 1-3, 6, 8-11, 14-17, and 19 is (TTTAGGG)_(n), the reverse complement, at 3′-ends. Examination of edges of assembly sequences from the algal genomes in Table 1 of the main text suggests Coccomyxa and Chlorella and possibly Monoraphidium are the same as Chromochloris, although Chlamydomonas appears to use (CCCTAAAA)_(n) and (TTTTAGGG)_(n). In Chromochloris, commonly observed non-canonical units are (CCTAAAA)_(n) and (CCCTGAA)_(n) near 5′-ends, and (TTTTAGG)_(n) and (TTCAGGG)_(n) near 3′-ends.

A prepared pool of Illumina reads was aligned with Bowtie2 in single end mode keeping top hit only to the ChrZofV5 assembly with PhiX; parameters were end-to-end “--sensitive” defaults, which allow short indels and up to ˜10% mismatches. Total pool nucleotides aligning to nuclear components was ˜26.8 Gnt, and the total size of pool members with ≥2 adjacent copies (not necessarily the same) of TAAACCC, TAAAACC, or TGAACCC or ≥2 adjacent copies (not necessarily the same) of AGGGTTT, AGGTTTT, or AGGGTTC was ˜62 Mnt. As the nuclear genome is ≈57 Mbp, this suggests Chromochloris telomeres total ≈133 Kbp (≈3.5 Kbp/end).

The beginning (relative to nominal ‘+’ strands) of chromosomes 5, 10-12, 14, and 16-17 and the end of chromosomes 4-5, 7, 12-13, and 18 were not reached in ChrZofV5. However, the presence of repeat units suggests that unplaced contigs chrUn97886, chrUn83064, chrUn12635, and chrUn01845 and possibly chrUn07087, chrUn06996, and chrUn06817 involve 5′-end telomeric junctions; and chrUn10942, chrUn10872, and chrUn03315 and possibly chrUn57207 involve 3′-end telomeric junctions.

Centromeres.

From experience with difficult sequence and gaps from Phases 3/4, candidate loci for centromeres (or, more likely, pericentromeric repetitive sequences surrounding them) were known for several chromosomes. For an unbiased scan, a visual examination was made of the whole genome distribution of each common TRF canonical tandem repeat unit. Focusing on units tending to concentrate in at most one zone per chromosome, iterative examination of sequence in and near these zones (by dotplots, BLASTing, local reassembly, and visualization of genome-wide occurrences) led to an expanding collection of putatively centromere-associated sequences; these were consistent with candidate locations. The collection converged on “ChrZofCen” (given later), a single circular ˜4 Kbp Type 1/Copia LTR retrotransposon with ˜0.7 Kbp spacer, together with TRF canonical units AAACATCTAG, AATCTGTGGTAGG, AAACATCTAGACACATCTAG, and AAACATCTAGACACATCTGG, with some 5S rDNA sequence.

TABLE S1 Start (‘+’ End (‘+’ Nominal Chrom. strand, bp) strand, bp) width (Kbp) Comments 1 ≈3,418,656 ≈3,457,392 ≈39 strong 2 2,093,247 2,141,774 49 strong, extra at 1,065,500-1,069,989 3 2,551,134 2,571,108 20 strong 4 2,648,641 2,651,949 3 possibly 2,719,643-2,722,347 (with asm. gap after) or 937,962-943,022 5 1,034,650 1,047,659 13 strong 6 709,495 716,341 7 strong, with assembly gap after 7 2,360,779 2,420,790 60 weak, with assembly gap inside 8 639,124 644,655 6 weak, with assembly gap after 9 in a gap in a gap ? no good candidates even though no large assembly gaps on this chrom. 10 860,629 862,963 2 weak 11 1,205,545 1,222,695 17 strong, with assembly gap inside 12 1,369,284 1,377,652 8 strong, with assembly gap inside 13 1,675,799 1,692,810 17 weak, with assembly gap inside; chromosome has large assembly gap 14 443,632 450,078 6 possibly 736,796-739,088 (with assembly gap before) 15 1,526,503 1,537,899 11 strong, with assembly gap before 16 490,261 510,147 20 strong, extra at 772,237-776,289 17 1,793,652 1,796,217 3 first option is at end of chrom., second or 126,717 or 127,771 or 1 with asm. gap after; 5 large asm. gaps 18 in a gap in a gap ? no good candidates; chromosome has three large assembly gaps 19 935,605 973,134 38 weak, with assembly gap inside

There are 39 unplaced contigs likely containing (peri)centromeric fragments:

chrUn{42003, 22516, 18154, 16591, 13058, 12366, 09183, 08437, 08040, 06312, 05306, 04914, 04275, 04018, 03492, 03384, 03059, 03028, 02729, 02724, 02655, 02649, 02593, 02484, 02398, 02352, 02284, 02246, 02034, 01939, 01933, 01883, 01678, 01641, 01499, 01429, 01415, 01238, 01183}. ChrZofCen (with IUPAC ambiguous nucleotides and ‘{option₁, option₂, . . . }’ curly braces capturing the most common variations observed) consists of the following coding portion (which, in all expansions, starts and ends on a codon boundary with ATG and TAA):

ATGACAGAACTGGAGAAGCTGGGTATCCCAArACTkAACGACCACAACTA TGTCTTCTGGCACATCAAGATGCGAGCCTACCTyGTTGCAAGAGGATACA GCGCAGCAATAACGAACGCAGAAGACGCCAACAGTGACAAGGCTCTTGCT TCCATCACTTTGGCTGTGGAAGATCATTTTCTACCTACAGTrTACAAwGC TGCAAGTGCGAAGGCAGCATGGGACGCGCTGGAGGCGTTGTTTCAGCAGC GGAGCGTTGCCAACCAGCTGAACCTCACGCAGGAACTGAACAACCTCACA CTGCAGCCTGGGGAGACCATCACACAGCTACTTGCTCGTGCCAGAATCAT ATGGGAGCAGCTTAAGGCAGCTGGTATCGACAAGTCAGAGCAGGAGGTGG CGTTATCAGTGTTGTCAGGACTTCCTGCCGACTTCAACACCTTAGTGACA GTACTACAGAATCAGTCTGGTCCmCTyACyCTGrGTGGCATCCAGAAGGC TGTCTTGACAGAACAGCAACGTGCAAATAAGGTTGGGGCATCAACGTCTA CTGCAGCAAGCACCAAGGCTTTCTACACTCAGAACGGTCCCAACCrTGGC ArGCTTGGTGACAGCGGTACCAGGACCAGCAACTT{,CAACCAGGGGAAC rG}CAACACCAAGCAGCAGGAGCAGCGTAAGTGCTACTACTGTGGCAAGA AGGGGCACCTGAAAAGGGACTGCAGAAAGAAGAAGGCAGACGAGCAGCGT GGCCCCAGTACCAAGGCTTCAACAACAATGGCATGGACTGCAGCCTGCAA CACCAGCATCAGCCTCAGCTCAGGTACCTGGGTCCTCGACTCTGGAGCAT CAAGACACGTCTGCAAAGAACGCAGCCTGATGCAGAACCTGCAACAGCTG AACCAGCCAGTCTACATCACGTACGGCAACGGTAGCACAGGGGTGGCACA GACTATGGGGGAGGTTGTTCTCAACGACAGGATCCGTCTACGGAACGTTT TGTTTGATCCCACTGCTGTTGGCAATCTCCTTTCCATCCsTACAGCAGCT GCryGTGGAGCACAGTTTAACTTTGsAGCCArTTGCTGCACCATTCGAGT AAATGGCAGACTGGTGGCAATAGCACAGCAGCAwGAyGGTCACTAyTGCT TGCACTCTGAGCAwrCAsAGTCAGCCACTGCACTGGCAGCCCAGACCCCG CAGCTGTGGCATCGTCGTTTTGGCCATCTCAGCTACCAGAATATGGCCAA GGTCCCCAACTTGGTAACGGGCGTCCAAGTsCCAACTGArGCCTTTCAGG CAGCAGGTCAGCAGGTGTGTGAGCCATGTCTACTkGGCAAACAGACACGA CTGTCTTTCCCCGAGTCAGArACTGTCAGGCAGCAGyCACTkGArCTGGT GCATATGGACCTCTGTGGACCTCTyCCTGTCAAGTCACTTGGAGGCAGCC AGTACATTGCTACGTTCCTGGAyGACTAyACAGGACTGTCAGTrGTGGCA TTGCTCAAACAGAAGTCAGACATTTCyAArGTTGTGCCTGACGTCTTCAA CATGCTAGAGAAACAGAGCAACAATCAGGTGAAGGGCGTCCGCACTGACA ACGGCGGGGAGTATGTCAACAATGTGmTGAACAGCTACTACAGCAGCAAG GGCATCATCGCACAGCACACAGTACCATACAGTCCTCAGCAGAATGGCAA GGCAGAAAGACTCAACCGAACCCTACTGGACAAGGCACGTTCCATGCTGG CAGATGCArGGCTACCTTCTCAGCTrTGGGGTGAGGCCGTGGTAACAGCC AATTATCTTAGGAACCGTTCACCAGCAGCTGGCAAGACAGCAACACCCTG GGAACTGTTTTTTGGGTCACGGCCCTCTGTCTCTCATCTTCGCGTGTTTG GGGCCAAGGCGTTTGCACAGATCCCCAAGGAGAAACGTGGCAAGCTGGAC CCAAGGAGTCAGCGTGGCATCATGGTTGGATATGAGCCyAATGTAAAGGG GTACCGTCTACTGCTTCCAAACAACACCATCACAGTCAGCCGGGACGTTG TATTTGATGAAGGTGACCAGCCAGGAGCArTAGACACCAACTTCTATCCA GACTTGGAAGATGAGCTTGATGTTACTGCAGCCATCAACACTGGATCTAA TGCAGCACCTTCTGTCAATACTTCTGGAACAGCTGAGCCACCACCATCAG TTGCAGCACCCGTCGACCCACCAATTTCGGCACAGACCATGGAAAACGTG GGAGCCAGCAACAGCTCAACACCACAAGGCAGyGAGGAAGATCAGCATCA GCAATCACGTAGAAGTAGCCGGGCCAACATTGGCATGGCACCAGGCAACT ACTGGGAGGCCAACTACATTCCCACATCCAAGCGTACAGCTACCGGACTG TTGGCACAGACATCAGAAATTGTTGAGCCAGCAACCTATGAmGAAGCACT ACAGTCAGACTGTGCAGAGCAGTGGCAGCAAGCCATGGACAGCGAGTACG CATCGCTGATAGCCAATGGAACTTGGACCTTGGAAAAACCCCCAACAGAC ATTAGGCCCATCCCTGTCAAGTGGGTGTATAAGGTGAAACGTGACACCAG CGGGAACATTGAGCGGTTCAAGGCACGCCTGGTGGCCAAGGGTTTTTGGC AACAGGAAGGTGTGGATTATGACGAAGTGTTCGCCCCGGTAAGCAAGTAT GCTACCTTTCGGGCACTAATGGCCAAGGCAGCAGAAGAGGACATGGAACT ACACAAATTGGATGTCAAGACTGCGTTCCTTCAAGGCAACCTGGAAGAAG ATGTTTGGATCCAGCAGCCTCGTGGCTACGAGGArGGCAGCAGTGAACTm GCCTGTCATCTwCAyAAACCTTTGTACGGGCTCAAGCAGGCyCCTCGrGC wTGGCATCAGCGGCTACAACAGGAACTACTGGCAGTAGGCTACACAGCAT CAGCAGCAGACCCCAGCCTGTACTGGTACTGCATCAACGGGGACTATGTG TACCTCCTGGTCTAyGTGGATGATATCCTGATTGCAGCCAAGCAGCTTGA GTCAGTCAAGGCAGTCAAGCAGCAGCTrTTAGGCTTATTTGAGTCGCGTG ACCTTGGAGAAGCwACATCCTACCTTGGTATGAGCATTCAGCGCAACAGA CAGACAGGCAyCATCAAGATyGGGCACCGACTCATGATCACAGAGTTACT GGArArGTATGGyGCAGTmGACAGCAAAAThAAGTCArTACCACTGTCTC CATCTATCAArCTrGCyAAAGATGAAGGCGryCCCCTAGACAAGGAACAT TACCCTTACAGCCAACTGGTTGGGAGTCTCATGTACCTTGCAATCACCTC CAGGCCAGACCTCGCCTTTTCTGTGGGGGCTCTTGCACGCTACATGTCAT GCCCAACCACwGTCCAyTGGCArGCAGCTAAGGGrGTrCTACGCTACTTG GGAGGAACCCTGGACTATGGCATCACCTTTGGTAGCGACAGCAATGACCT CATTGGCTACTGTGACGCAGACTATGCGGGAGACACAGACACACGCAAGT CCACCAGTGGCTACATATTCATACTGCACGGAGGGGCCATyACkTGGAGT AGTAAGCGCCAGGCAACAGTTGCAGCmTCAACCACGGAGGCTGAGTACAT GGCAGCAGCAGCAGCAGTCAAGGAAGCTCTATGGCTGCGTACACTCTTGA GCGAGCTGCAGCTAGACATAGACAACATCACTATCATGGCAGACAACCAG TCAGCAATCAAGCTTCTGCGCAATCCTATCTCATCCATGAGAACCAAGCA CATTGAyGTGGCTTATCACTTTGCTAGGGAACGCGTGGTGCGCAAGGAGG TTGTGTTCAGGTTCGTTTCCACAGAGAACATGGTGGCAGACATCATGACC AAGGCTCTGAGCGAAGTCAAGCATGTGCGATGTTGCAAGGGCATGGGGGT TGGAGTTTAA followed by a more variable spacer region

AGAAACTTGAAATGCGTGGGAGCATCTTTGACAGTACATGCCTGACTGC GTGGGAGTGTTGAAATACGGCCTTTATTCAGTCAGACCTGCACTGCCAG AATCCAGAAGTTGAGCATCTTTGACAGTACATGCCTGACTGCGTGGGAG TGTTGAAATACGGCCTTTATTCAGTCAGACCTGCACTGCCAGAATCCAG AAGTTTCCAGATGGTTCTGGAAGyCCCCAGATGTTTCyAGATGTTTCyA rATGTkTCy{,AAATGTGTCC}{,AGAAGTTTCTAGAGGTGTCTAGATG TTTCT}AGAATATTGGTGCATGACACGTGTCAGTCACTTTGTGGTr{,G TAGGAATCTGTG,GTAGGAATCTGTGGTAGGAATCTGTG}GTAGGAATC TGTGGTAGGAATCTGTGGTAGGAATCTGTGGTAGGATTCCCAGTAGGTG AACACAGTTGCCAGTGGATTGCCATTGTGTCGTGAGTATATAAAGACAC AGACTTGTCCCAATCTGTAAyAyTGTCCAGCCCGAGysCCACCGAGGCC CCACGCTTAAACACAGACCGCAACACAGAGCTGAGGATACTGAGTCGCT AGAACGACTwAGrCAACAGATTTCCATCAGGTTATGGGCCCACrCCCAC ACGCACAATCGCTGTGCTGCTCAGAAATTTGTTGTGTTCGGCCATAAGT GTTGTGTACAGTTCGTCArCmAGGTCACd which then circles back to the beginning of the coding region. The Type 1/Copia LTR retro-transposal nature is clear from NCBI web conserved domain hits to its amino acid translation, these being DUF4219 (a domain associated with the N-terminus of gag-pol proteins), UBN2 (gag of LTR Copia type), ZnF_C2HC/zf-CCHC (zinc knuckle associated with retroviral gag), gag_pre-integrs (part of gag lying just upstream of integrase), rye (the integrase core domain), RVT_2 (reverse transcriptase), and RNase_HI_RT_Ty1 (RNase H for Type I/Copia LTR retroelements), in that order. NCBI web BLASTX had best hit to filamentous green alga Klebsormidium flaccidum with second best organism being colonial green alga Volvox carteri.

Due to the difficulty of assembling such large-unit repetitive sequence occurring in multiple tandem arrays (and reads suggest each array consists of complex nested insertions of mixed orientation with some divergence), ChrZofV5 in (peri)centromeres—even when given as gapless pure A/C/G/Ts—may have considerable errors. Almost all putative (peri)centromeric intervals given in the table on the previous page are associated with major assembly gaps and/or fine size differences between in silico BamHI fragment lengths and the optical map (with the assembly generally being too small; see also the discussion later about known assembly problems). However, borders and entry into pericentromeric sequences should be of quality comparable to the assembly generically, the optical map prevents massive errors (and constrains sizes), and sequence presently in ChrZofV5 should be representative. An estimate of the total size of (peri)centromeres was obtained in two ways. First, there is ≈195 Kbp of N-free sequence in the called intervals of the table and ≈231 Kbp of N-free sequence in the identified unplaced contigs/scaffolds (although all of such may not belong, as edges may be ordinary nuclear sequence), a total of ≤426 Kbp. Second, an analysis similar to that using Bowtie2 for the telomeric sequences (except selecting prepared Illumina reads as those having at least one 19-mer hit to either strand of all IUPAC-ambiguity and curly brace expansions of ChrZofCen or any rotation of (AAACATCTAG)₂, (AATCTGTGGTAGG)₂, AAACATCTAGACACATCTAG, or AAACATCTAGACACATCTGG) finds ≈290 Mnt of (peri)centromeric reads vs. the ˜26.8 Gnt of nuclear reads, suggesting total centromeric nuclear sequence of ≈618 Kbp. Thus, Chromochloris may have a total of ≈0.5 Mbp of (peri)centromere, an average of ≈25 Kbp per chromosome.

Ribosomal DNA (rDNA).

The canonical rDNA repeat unit for Chromochloris became apparent early in assembly during analysis of k-mers observed with high frequency, and is given as contig chrRr. It assembled as a 9,702 bp circular consensus which RNAmmer (48) annotates as follows (the consensus was oriented so that annotations fall on the ‘+’ strand, and the de-circularizing linearization cut was placed just before the 28S rRNA annotation):

Typically, rDNA exists in at least one large tandem array; such sequence is, however, difficult to assemble. As with the (peri)centromeres, bordering sequence and entry into such regions is expected to be less problematic. From the plot, the tail of chromosome 13 (relative to its ‘+’ strand) leads into a large assembly gap with rDNA sequence at the left border (indicated by purple oval). Further, the rDNA consensus contains two BamHI sites which, in circular form, produce fragments of sizes ˜6.0 Kbp and ˜3.7 Kbp that are both in the range in which the optical mapping worked well, and this region of chromosome 13 has an optical tandem repeat of copies of an alternation of fragments ≈6 Kbp and ≈4 Kbp in size. This suggests that the large assembly gap at the end of chromosome 13, estimated to be ≈593 Kbp in size, begins with ≈24× copies of the rDNA unit (likely with divergence among copies); these copies would represent ≈233 Kbp or ≈40% of the gap. Various analyses (e.g., that of Table 1 in the main text) assume this gap begins with 24× exact copies of chrRr.

Repetitive Sequence.

There are repetitive sequences beyond the telomeres, (peri)centromeres, and rDNA already discussed. The nuclear fraction of the ChrZofV5 assembly was analyzed with RepeatMasker 4.0.6open (using slow search and gccalc options with engine RMBlast+2.2.28) in combination with all of Repbase Update 2016 Aug. 29 (“eukaryota”) and de novo identified repeats from RepeatModeler 1.0.8open with RepeatScout 1.0.5, RECON 1.08, TRF 4.04, and RMBlast+2.2.28 (SI File S8). About 6% of the assembly (excluding N-runs) was masked, mostly in interspersed repeats (˜5.0% of sequence) as primarily LINEs (˜2.0%), LTRs (˜1.5%), unclassified elements (˜1.2%), and DNA elements (˜0.4%). The remainder was mostly simple repeats (˜1.0%), with some satellites, low complexity sequence, and small RNA (total ˜0.1%).

Per chromosome plots of repeat and gene density were prepared. Gene density is rather uniform, and there are no grand scale gradients in genes or repeats as found in, e.g., Arabidopsis, a genome approximately twice as large that has megabasepairs of pericentromeric heterochromatin (49). Some smaller scale gradients in repeats are found near (peri)centromeres and especially large assembly gaps (e.g., the large gaps of chromosomes 17 and 18). There are a few localized concentrations of particular kinds of repeats. As apparent from Table 1 of the main text, Chromochloris, like Coccomyxa, has relatively few repeats compared to other algal genomes of comparable size (Chlorella or Monoraphidium) and much fewer than larger genomes (Chlamydomonas or Arabidopsis).

Known Assembly Issues.

From the hand word and detailed comparison of the final ChrZofV5 assembly to the optical map, 100 areas where the assembly has issues are known. (These are in addition to a likely number of very localized errors, e.g., individual basepairs; assembly polishing by variant detection using re-aligned reads is pending for the next assembly release.) About half of issues (52/100) are represented in the ChrZofV5 assembly by runs of one or more N bases (typically with length a multiple of 1,000 bp sized approximately correct via the optical map); another half (47/100) are deviations in number of BamHI fragments or fragment lengths between the assembled sequence and optical map beyond norms; and a final one (1/100) is the optically-troubled tail of chromosome 5 mentioned earlier. Issues are detailed in SI File S4 and summarized in SI File S5. Below is a brief discussion of the summary and largest issues.

The largest assembly gaps (≥≈100 Kbp) are: one in the interior of chr. 4 (issue V5.04.4) of ≈193 Kbp; one at the beginning of chr. 11 (V5.11.1) of ≈107 Kbp; one at the end of chr. 13 (V5.13.6) of ≈593 Kbp of which, as already discussed, the first ≈220 Kbp of which is likely ≈24× copies of the rDNA repeat unit (so the amount missing is more like ≈373 Kbp); one in the interior of chr. 14 (V5.14.2) of ≈128 Kbp; three in the interior of chr. 17 (V5.17.2, 0.4, and 0.6) of ≈120, ≈295, and ≈138 Kbp; and two in the interior of chr. 18 (V5.18.1 and 0.2) of ≈248 and ≈208 Kbp. Chromosomes 17, 18, and 13 have by far the most assembly gap as a proportion of optical length. The presented sequence of chromosomes 1, 2, 3, and 9 is gapless, although, as discussed earlier under, e.g., centromeres, this does not imply they are perfect. Much of the “missing” sequence is expected to be among the unplaced contigs/scaffolds.

OpGen did not observe the right end of chromosome 5 (issue V5.05.10); its map ends in an inverted optical repeat of ≈150 Kbp per arm. From patterns of chromosomal coverage by reads and detailed hand examination of Illumina and PacBio reads aligning and partially aligning in this region, it was determined after the ChrZofV5 assembly was frozen and this publication was initially submitted that the likely resolution is this inverted repeat is much larger—fully ˜564 Kbp per arm—with the right arm exiting directly into telomere repeats. Using 1-based inclusive-inclusive ‘+’ strand coordinates in ChrZofV5, a full left arm is given as chr05:3230407-3794116 and a full spacer between the two arms is given as chr05:3794117-3795139, but the end chr05:3795140-3801251 only gives ˜6 Kbp of a right arm (and with chr05:3796510-3798042 being naked single-read PacBio sequence). A quick patch is to tack revComp(chr05:3230407-3788934): AAGGGTTTAGGGTTTAGGGTTTAGGGTTTAGGGTTTAGG GTTTAGGGTTTAGGGTTTAGGGTTTAGGGTTTAGG onto the end of ChrZofV5 chr05, making chr05 longer by 558,602 bp, but it is planned for the next genome release to use the PacBio reads to phase the two arms (which appear to have some variation) and give a better representation (as the sequence currently in ChrZofV5 is presumably randomly phased). Note there are 155 current gene models affected (Cz05g32080, . . . , Cz05g37220 in the left arm and Cz05g37230 and Cz05g37240 in the right); there will be another ˜153 once the rest of the right arm is added. Except for this paragraph, this disclosure assumes a genome with most of the right arm absent.

ChrZofV5 chromosomes 1 to 19 total 57,719,290 bp (including N placeholders); the optical map totals 57,763,775 bp (with only the first half of the chromosome 5 optical repeat counted). These agree to ≈1 part per thousand and, when quoting lengths as fractions of the nuclear genome, it does not matter much which is taken as reference whole. (The total differs by under 45 Kbp and single chromosomes by under ±42 Kbp.) About 5% of total (=3 Mbp) is missing over the 52 runs of Ns; the unplaced contigs/scaffolds presumably provide ≈2.4 Mbp of this (≈80%). Over the 47 BamHI fragment disagreement issues, assembled sequences are estimated to be missing ≈512 Kbp and have ≈45 Kbp extra; this is under 1% of total and a smaller class of problem than the runs of Ns. Thus, ≈6% of total is missing or otherwise troubled, but ≈94% is placed and in tight agreement with the optical map. Although current data is not exhausted and additional refinements can be made, in the interest of timely availability to the community of the already high quality genome, the ChrZofV5 version is being publically released.

Genomes of the Chloroplast and Mitochondrion

Assemblies of organelles took place between nuclear assembly phases and also required multiple hand-managed passes (as they were not assembled whole by any of the automatic processes). Various methods were used to identify potentially relevant contigs and reads, including relatively high coverage, low G+C content, alignments and synteny to existing NCBI chloroplast and mitochondrion sequences, and alignments to seed contigs once some were in hand.

Mitochondrion.

The mitochondrion (for SAG 211-14, the strain of this study) was completely assembled as a single circular 41,733 bp contig chrMt with no IUPAC ambiguous nucleotides; the strand orientation and linearizing cut were chosen to agree with NCBI accession KJ806268.1, the 44,840 bp complete mitochondrion of Chromochloris zofingiensis strain UTEX 56. Annotation of protein-coding genes, tRNAs, and rRNAs of chrMt was by BLASTN/BLASTX and BLASTP to the NCBI ‘nt’ and ‘nr’ databases, tRNAscan-SE, RNAmmer, Rfam, syntenic alignments to closely related known sequences (e.g., to KJ806268.1), and visual examination of RNA-Seq alignments (which suggest some UTRs, although these were not kept in the final annotations). The overall structure of chrMt is highly similar to KJ806268.1, having the same major protein-coding genes, tRNAs, and rRNAs in the same order, however there is considerable divergence at the nucleotide level with a global pairwise alignment (Geneious 93% similarity cost matrix, gap open penalty 30, gap extension penalty 1; only ˜66% identical. Divergence is concentrated intergenically and the splicing structure of rrnL4 is different. Globally aligning just the coding sequences results in ˜98% nucleotide identity. Translating the coding sequences via NCBI genetic code #22 (the Scenedesmus obliquus Mitochondrial Code) and globally aligning (Geneious BLOSUM62, gap open penalty 12, gap extension penalty 3) estimates ˜99% amino acid identity.

Chloroplast.

Similarly, the chloroplast (for strain SAG 211-14) was completely assembled as a single circular 181,058 bp contig chrCp, also with no IUPAC ambiguous nucleotides; the strand orientation and linearizing cut were chosen to be in agreement with NCBI accession KT199251.1, the 188,935 bp complete chloroplast of C. zofingiensis strain UTEX 56. Again, annotation of protein-coding genes, tRNAs, and rRNAs was by BLASTN/BLASTX and BLASTP to NCBI ‘nt’/‘nr’, tRNAscan-SE, RNAmmer, Rfam, syntenic alignments to closely related known sequences, and visual examination of RNA-Seq alignments (which again suggested some UTRs, although as before these were not kept in the final annotations). As with many chloroplast genomes, there is a large rRNA-related inverted repeat (˜6.7 Kbp in SAG 211-14, ˜6.4 Kbp in UTEX 56) separating two single copy regions. It is difficult to resolve the arms with short reads; they assembled as identical except for a tandem repeat CTTGGTATTGGGGC estimated as 8× in the first arm and 9× in the second (where SAG 211-14 inserts ≈300 bp relative to UTEX 56). The relative strand orientation of the single copy regions is ambiguous, and no PacBio reads were found able to resolve this. The single copy regions were assembled in opposite relative strand orientation compared to KT199251.1, and so in further comparisons the second single copy region of KT199251.1 was reverse complemented.

With the second single copy region of KT199251.1 reverse complemented, the overall structure of chrCp is highly similar to KT199251.1, with the major protein-coding genes, tRNAs, and rRNAs again in the same order. Aligning in the same way as with the mitochondrial genomes, global alignment gives overall nucleotide identity of ˜83% and global alignment after restriction to coding sequences gives ˜98%; divergence is again concentrated intergenically. The largest difference is the loss in SAG 211-14 of almost all of a ˜9.3 Kbp region in UTEX 56 annotated as containing a ptz-like ORF. Translating the coding sequences via NCBI genetic code #11 (the Bacterial, Archaeal, and Plant Plastid Code) and globally aligning results in ˜97% amino acid identity, with lower percent identity in the larger genes (e.g., ftsH, rpoC2, and ycf1). The gene psaA remains trans-spliced (with RNA-Seq in concurrence); an in silico effort to identify a homolog of the Chlamydomonas tscA gene involved in this process was unsuccessful.

From the Bowtie2-based analysis introduced in the telomere discussion earlier, coverage on chrMt and chrCp from prepared Illumina reads is ≈1,280×(≈0.2% of sequencing effort) and ≈890×(≈0.6%), respectively. Coverage of PhiX is ≈150,000×(≈2.8%), and the nuclear genome (chromosomes, rDNA, and unplaceds) is ≈460× on average (293.9%). The remaining ≈2.6% of effort is in reads that did not align; ≈1.5% is accounted for in a re-alignment to Illumina inline controls, leaving ≈1.1% of effort unaligned. At nuclear coverage, this could be ≈0.7 Mbp of additional sequence, very close to the ≈0.6 Mbp more expected beyond current unplaced contigs/scaffolds as discussed under “Known assembly issues” above. The high fraction (≈98.9%) of original reads accounted for is encouraging; there is not much sequence missing from the ChrZofV5 assembly, even if it is not yet in perfectly contiguous form.

Transcriptome Assembly Used in Training AUGUSTUS

To assist with training the AUGUSTUS ab initio gene modeler for Chromochloris, a draft transcriptome was de novo assembled from the 151+151 nt pool of ˜397M read pairs from the fourteen RNA-Seq sub-libraries described earlier, using the Ray assembler with k=51. Such de novo transcriptome contigs are generally presented in random strand and codon frame, generally contain UTRs, and may contain introns (and many of large number of shorter, lower-coverage contigs may be wholly introns, and introns may change codon frame). To bootstrap AUGUSTUS, PASA 2.0.2 was used to extract a training set of genes (50).

Details of Main Text Table 1

Nuclear Genomes.

Sequences and annotations (especially those of model organisms) are often updated after initial publication, and details of definitions and statistical analyses can often greatly affect summaries. For these reasons, Table 1 was completed by analyzing freshly-downloaded current copies of reference genome sequences and gene models and uniformly applying the same criteria and methods to every organism rather than, e.g., copying nominal quantities from existing publications. Sources of nuclear genomes and annotations were TAIR10 for Arabidopsis thaliana (“AraTha”), JGI Phytozome 5.5 for Chlamydomonas reinhardtii (“ChlRei”), ChrZofV5 of this work (with 24× copies of the rDNA unit) for Chromochloris zofingiensis (“ChrZof”), JGI Phytozome 2.0 for Coccomyxa subellipsoidea C-169 (“CocSub”), JGI release 2014 Aug. 18 with ‘best genes’ for Chlorella sp. NC64A (“Chlore”), and NCBI accessions KK100223.1-KK106940.1 for Monoraphidium neglectum (“MonNeg”).

Sequenced Genome Size:

number of non-N/n bases in assembly (other IUPAC ambiguities were retained), rounded to nearest Mbp. Sequenced genome presentation: a “scaffold” is defined as a nucleotide sequence having at least one N/n (with other IUPAC ambiguities being irrelevant) and presuming every other sequence to be a “contig”. For CocSub, all sequences are called “scaffolds” in distributed files and chromosome vs. arm vs. unplaced is not indicated; however, the distinctions are clear from presence of telomere-associated repeats at one, both, or neither sequence edge, and the number of chromosomes plus half the number of arms as thus determined equals the stated 20 chromosomes in the associated genome paper (51), which also mentions that, via Southerns, the pairing of half the arms was determined. Genome project primary initial strategy, average basepair coverage at earliest stage: per best evidence available and literature, including CocSub (51), AraTha (49), ChlRei (52), Chlore (53), and MonNeg (54).

Scaffold N50 (Taking Genome Size as Sum of Scaffolds as-are):

ordering scaffolds by decreasing size (and keeping all IUPAC ambiguous nucleotides), take scaffolds until total size is at least as large as half total size of all scaffolds, and report size of the smallest taken scaffold after rounding to the nearest Kbp. This was not performed for assemblies at chromosome/arm scale, as this quantity is then essentially as large as it can be and is controlled by the organism's distribution of chromosome sizes and is no longer connected to assembly quality. Contig N50 (taking genome size as sum of contigs as-are): form “contigs” by splitting scaffolds at every N/n (tolerating other IUPAC ambiguities) and removing all N/ns; order contigs by decreasing size, take contigs until total size is at least as large as half total size of all contigs, and report size of the smallest taken contig after rounding to the nearest Kbp. Number of chromosomes: per best evidence available and literature. For Chlore, although not mentioned in the associated genome publication (53), the largest scaffolds are large and one can look for telomere-associated repeats; 11 of their scaffolds begin with such a repeat and 7 end with one (and none have both ends thus associated), which is more or less consistent with the genome publication's determination of 12 chromosomes by Pulsed-Field Gel Electrophoresis (PFGE), with chr. 12 being difficult.

Protein-coding genes are taken as those directly declared as such by the annotations; in cases (MonNeg) without a direct indication, a GFF file gene was taken as protein-coding if and only if it had non-empty intersection with at least one GFF file CDS interval. Three of the releases here (for ChrZof, MonNeg, and CocSub) do not provide multiple transcript models per gene locus. (Although the CocSub release includes versions of files named so as to distinguish all models vs. “primary transcript only”, such versions are the same and no protein-coding locus is actually modeled with multiple isoforms.) For AraTha, when desired, the canonical transcript model for each gene locus is per TAIR's file TAIR10_representative_gene_models.gz. For ChlRei, when desired, the canonical model is that marked longest=1 in the annotation GFF files (and all GFF files in the release agree on this designation). For Chlore, there is no issue since the only gene models used in this work are those from the release's ‘best genes’ files. Note that MonNeg is a highly fragmented assembly and one may expect (in agreement with the BUSCO analysis of main text FIG. 2B) its gene models to suffer due to, e.g., true coding sequences often reaching edges of assembly sequences; for this reason, numerous of its gene-related summary statistics may be more divergent from “truth” than for the other organisms.

Percent G+C in Sequenced Genome:

using only A/C/G/i nucleotides (no IUPAC ambiguities) in an all-uppercase version of the assembly, report fraction (# C+G)/(# A+C+G+T) as a percent, rounded to the nearest integer. Basepairs called as coding (in any transcript model) in sequenced genome: over all transcript models of all protein-coding genes, take union of coding sequence bases (ignoring strands) to get a target subset of assembly basepairs; restrict to the N/n-free fraction of this subset and the whole assembly (other IUPAC ambiguities being tolerated), and report percentage of the whole in the subset after rounding to the nearest integer. Percent G+C in basepairs called as coding (in any transcript model): over all transcript models of all protein-coding genes, take union of coding sequence bases (ignoring strands) to get a target subset of assembly basepairs; using only A/C/G/i bases (no IUPAC ambiguities) in an all-uppercase version, report fraction (# C+G)/(# A+C+G+T) as a percent, rounded to the nearest integer. Number of “complete” called protein-coding gene loci (collapsing transcripts): same as the row “Number of called protein-coding gene loci (collapsing transcript forms)” except restricted to coding sequences that satisfy all of the following: (1) are pure A/C/G/T (i.e., contain no IUPAC ambiguities); (2) have length a multiple of three nucleotides; (3) start with ATG; (4) end with TAA/TAG/TGA; and (5) do not contain an internal TAA/TAG/TGA codon.

Number of rDNA Units Estimated to Exist in True Monoploid Genome:

MonNeg and AraTha are via (54) and (55); for ChrZof, this work as, e.g., already described in subsection Ribosomal DNA (rDNA). For ChlRei, the original genome paper (52) contains some information but not quantitation. Seven paired-end 76+76 nt Illumina GA-II lanes of a Chlamydomonas genomic library were available from an unrelated project. Extremely high coverage 39-mers from the reads were de novo assembled, rDNA-related seed contigs selected via NCBI web BLASTN, paired-end reads having at least one 31-mer from the seed contigs and seen multiple times were extracted and re-de novo assembled to obtain a 6,543 bp consensus chunk of a presumed Chlamydomonas rDNA unit. The chunk contains a whole 18S followed by a whole 28S. Comparison of median Jellyfish 39-mer coverages for the consensus chunk of rDNA unit vs. some generic “1×” ordinary sequence in the nuclear genome (that N/n-free chunk of chr. 1 with length 440,320 bp, with a coverage threshold to remove empirically non-unique regions) provides an estimate of rDNA unit copy number as 840× (independent of the chunk's tandem circle not being closed), and ˜5.5 Mbp as a lower bound on total length (dependent on the fraction of the unit the chunk represents).

Number of tRNAs Called in Sequenced Genome:

counts are for all types (including with introns, unclassified, selenocysteine, and pseudo). For AraTha, the TAIR release contains explicit tRNA annotations, and the table entry ‘631’ counts these. For the other organisms, even though, e.g., the original genome papers generally discuss tRNAs (implying that predictions were made), the annotation releases do not identify tRNAs and so for this work ab initio scans with tRNAscan-SE 1.3.1 were performed with default parameters. (This scan finds 639 for AraTha.) For MonNeg, the ab initio scan found 38 in the nuclear genome, 29 in the chloroplast, and 23 in the mitochondrion, while the original genome paper (54) states “40+1× Pseudo Ser-tRNA” for nuclear, “29+1×Pseudo Leu-tRNA” for the chloroplast, and “21+1×Pseudo Met-tRNA” for the mitochondrion in its Table 3 but shows 23 in its FIG. 5. For ChlRei, the ‘259’ shown in Table 1 is taken from the original genome paper (52), as even though the current JGI 5.5 release does not contain tRNA annotations, the original genome paper states that tRNAscan-SE is known to overestimate in Chlamydomonas due to tRNA-associated SINE retrotransposon elements; the ab initio scan predicts 353 tRNAs in the current nuclear assembly. Regarding ChrZof, the scan only identifies 75 tRNAs (SI File S9)—more than Chlore and MonNeg, the small algal genomes of high G+C and moderate repeat content, and similar to CocSub, the other algal genome of moderate G+C and low repeat content, but much less than the relatively large genomes of AraTha and ChlRei; there are no large clusters, although there are runs of up to four on the same chromosome with spacing smaller than would be expected at random (e.g., with some adjacencies closer than 1 Kbp).

TABLE S2 # # # missing Organism: selenocysteine: pseudo: undetermined: std. AAs: CocSub 0 3 4 none ChrZof 0 0 0 none AraTha (ab initio) 0 8 1 none ChlRei (ab initio) 1 2 2 none Chlore 1 0 0 Ile MonNeg 2 1 1 Asn, Glu, Trp, Tyr

From the ab initio scans, all standard amino acids are covered for all six organisms, except for one in Chlore and four in MonNeg, perhaps because these are the most fragmented assemblies, or perhaps due to tRNAscan-SE misclassifications as selenocysteine/pseudo/undetermined (of which there are exactly one and four in Chlore and MonNeg, respectively). The phylogenetic profile of anticodons (ignoring predicted pseudogene status) is as follows: universal in all six=AGC, AGG, AGT, CAA, CAC, CAT, CGC, CTG, CTT, GAA, GCA, GCC, GTC, GTG, TCG; missing from all six=AAA, ACA, ACT, ATA, ATG, ATT, CTA, GAC, GCG, GGC, GGG, GGT, TTA; missing from just MonNeg=AAC, AAG, ACG, CAG, CCA, CGA, CGG, CTC, GTA, GTT, TAA, TGG; missing from MonNeg and Chlore=AAT, CCG, CCT, TAC, TCC, TGA, TGC, TGT, TTC, TTG; missing from just Chlore=CCC, CGT, GCT, TAG, TAT, TCT; missing from MonNeg, Chlore, and ChrZof=AGA, TTT; just AraTha=ACC, GAG; just ChlRei=ATC; just MonNeg=GAT; just AraTha and CocSub=GGA; and just ChlRei, Chlore, and MonNeg=TCA.

Number of Amino Acids: {Average, Median}:

gene models are taken without question (e.g., even if one does not start with a start codon, end with a stop codon, has coding sequence not a multiple of three nucleotides in length, the coding sequence contains IUPAC ambiguities, the coding sequence is very long) and the result rounded to the nearest integer. Number of exons containing coding sequence: {average, median}: gene models are taken without question (e.g., no matter how many exons they have) and the result rounded to the nearest tenth. Exon length (restricted to coding sequence): {average, median}; Intron length (between exons with coding sequence): {average, median}; Percentage with at least one intron (between exons with coding sequence): same comments as for “Number of amino acids: average”.

% of Seq. Basepairs RepeatMasker'd with {Repbase Update “Eukaryotic”, RepeatModeler, RepeatModeler+Repbase Update “Eukaryotic”}:

the RepeatMasker/Repbase/RepeatModeler analysis discussed earlier for ChrZof under subsection Repetitive sequence was applied to the other five organisms with the same parameters. Masking was variously with just known repeats (Repbase only), just de novo repeats from RepeatModeler, and the combination of the two.

Chloroplasts.

For chloroplast genomes, reference sequences and annotations were as follows. CocSub: NCBI accession NC_015084.1 (with one sequence gap and lacking a large inverted repeat) and annotations. ChrZof: chrCp of the ChrZofV5 release of the present work. AraTha: NCBI accession AP000423.1 sequence and annotations. ChlRei: NCBI accession FJ423446.1 sequence and annotations. Chlore: NCBI accession KP271969.1 sequence (lacking a large inverted repeat) and annotations. MonNeg: NCBI accession CM002678.1 sequence, but with annotations from FIG. 4 of the original genome paper (54) as the annotations deposited at NCBI are manifestly highly incomplete. Sequenced genome size. The number of non-N/n bases in the assembly (other IUPAC ambiguities being tolerated) is reported, rounded to the nearest Kbp. Number of annotated protein-coding genes, including hypotheticals; Number of annotated {rRNAs, tRNAs}: if the genome contains large repeats (as common in chloroplasts), genes are counted as +1 copy for each copy of the parent repeat. For tRNAs, as with the nuclear genome, if no annotations were provided, an ab initio tRNAscan-SE scan was performed (and all types counted). Percent G+C in sequenced genome: using only A/C/G/T bases (no IUPAC ambiguities) in an all-uppercase version of the assembly, the fraction (# C+G)/(# A+C+G+T) is reported as a percentage rounded to the nearest integer.

Mitochondria.

For mitochondrial genomes, reference sequences and annotations were as follows. CocSub: NCBI accession NC_015316.1 sequence and annotations. ChrZof: chrMt of the ChrZofV5 release of the present work. AraTha: NCBI accession JF729201.1 sequence and annotations. ChlRei: NCBI accession NC_001638.1 sequence and annotations. Chlore: NCBI accession NC_025413.1 sequence and annotations. MonNeg: NCBI accession CM002677.1 (with two sequence gaps) and annotations. Rows are the same as for chloroplasts, except for the following note not already mentioned elsewhere: the MonNeg mitochondrial sequence has no rRNA annotations (and two sequence gaps); RNAmmer does not find any rDNA, but Rfam finds four zones with LSU/SSU fragments.

Calling of Protein-Coding Gene Families Across the Six Organisms of Main Text Table 1

To call gene families simultaneously across AraTha, ChlRei, ChrZof, CocSub, Chlore, and MonNeg (the six organisms of Table 1 of the main text), the amino acid sequences of the genes corresponding to row “Number of called protein-coding gene loci (collapsing transcript forms)” of Table 1 were collected. Alignment seeds were formed by running NCBI BLASTP+2.4.0 with E-value threshold 10⁻⁵ and soft masking (segmasker window 12, locut 2.2, hicut 2.5) on both queries and subjects (and otherwise defaults, including BLOSUM62 scoring). For every distinct ordered pair (query, subject) with at least one BLASTP+result, global Needleman-Wunsch alignment was performed with the C++ library Parasail (BLOSUM62 scoring with gap open and extend penalties 10 and 1, respectively). Compared to the local alignments of BLASTP, the global alignment score captures not only sequence similarity, but also aspects of the fraction of the entirety of query and subject aligned and the ordering of homologous fragments (e.g., component protein domains).

In the first phase, “self-prefamilies” were formed within each organism. For Parasail-aligned pairs of genes (query, subject=query) with global alignment score s≥16, keep as “tentative arcs” those Parasail pairs (query, subject in same organism except query itself) with global alignment score ≥85% of s. Remove tentative arcs (query, subject) for which (subject, query) is not a tentative arc, so as to obtain unordered pairs {gene, different gene in same organism} that constitute edges in an undirected graph. Partition vertices of this graph (the pieces of this partition being the self-prefamilies) by subdividing the vertices of each connected component as follows: (1) find all maximal cliques in the connected component; (2) keep only cliques of maximum size; (3) expand each clique to also contain those vertices in the connected component that are adjacent to at least half the vertices in the clique; (4) keep only expanded cliques of maximum size by number of vertices in them; (5) group vertices in the union of the surviving expanded cliques by their combination of membership status in the surviving expanded cliques, these groups becoming pieces of the final partition; and (6) recurse [going back to (1)] on any vertices remaining. Finally, each gene in the organism not represented is added as a singleton self-prefamily (of size 1). Self-prefamilies involve 1 to 31 genes (but only 1 to 4 genes each when restricting to sizes occuring ≥10 times in any single organism, and only 1 or 2 genes each when restricting to sizes seen ≥100 times in any single organism). The percent of genes in self-prefamilies of size ≥2 is ˜8.4% and ˜4.6% in the large genomes AraTha and ChlRei, respectively; ˜2.9% and ˜2.2% in the algal moderate G+C content genomes CocSub and ChrZof of low repetitive sequence fraction, respectively; and ˜1.1% and ˜0.9% in the algal high G+C genomes Chlore and MonNeg of moderate repetitive sequence fraction, respectively.

Self-prefamilies exhibit evidence of tandem duplication events in all six genomes. For example, consider self-prefamilies of size exactly 2. (Across organisms, this is ˜73% to ˜96% of self-prefamilies of size ≥2.) Given such a self-prefamily, classify it as type “Far” if the two genes are on different sequences in the reference genome or the midpoint of the bounds of their coding sequences are ≥20 Kbp apart; otherwise, classify it as type “Near⁺” if the two genes are on the same strand or “Near⁻” if they are on opposite strands. There is enrichment for Near⁻ and larger enrichment for Near⁺ in every organism:

TABLE S3 # observed: random expectation: observed/expected: Organism: Far Near⁺ Near⁻ Far Near⁺ Near⁻ Far Near⁺ Near⁻ AraTha 714 214 30 ~958 ~0.181 ~0.170 ~0.75 ~1,184 ~176 ChlRei 146 58 30 234 0.040 0.041 0.62 1,454 725 Chlore 20 16 6 42 0.017 0.018 0.48 943 328 ChrZof 104 12 8 124 0.040 0.040 0.84 298 199 CocSub 41 13 1 55 0.020 0.023 0.75 649 44 MonNeg 59 9 1 69 0.006 0.006 0.86 1,512 174

In the second phase, “prefamilies” are formed—these target orthologs (“primaries”) and generally involve more than one organism. For Parasail-aligned pairs (query, subject in different organism) sharing the same query, drop all these pairs if the best global alignment score s is <16 and otherwise keep only pairs with global alignment score ≥97% of s. Replace kept ordered pairs of genes (query, subject) with ordered pairs (self-prefamily of query, self-prefamily of subject) and thin ordered pairs seen more than once down to a single copy. Taking these as the new “tentative arcs”, follow the same procedure as used to form self-prefamilies, except the resulting partition pieces now constitute the prefamilies. Each of these involves 1 to 15 self-prefamilies; ˜67% and ˜90% of genes in multi-organism prefamilies belong to prefamilies with at most 1 and at most 2, respectively, self-prefamilies per organism.

In the third phase, final families are formed—with paralogs now also targeted as “additional” genes in each family—by merging into each multi-organism prefamily zero or more single-organism prefamilies. Each single-organism prefamily S is considered independently one at a time: for each gene a in S, gather Parasail alignments (a, gene b in a multi-organism prefamily) and (gene b in a multi-organism prefamily, a), keep only alignments with maximum global aligment score, and note the multi-organism prefamilies that surviving b belong to; if exactly one multi-organism prefamily M is noted after all a are considered and at least one kept alignment was seen with strictly positive global alignment score, then S is merged into M as additional genes (and otherwise S is left alone). 5,258 multi-organism prefamilies receive merges, each 1 to 196 times, with ˜88% of these ≤6 times. There are 41,328 final families (these partitioning all 27,206+17,741+15,344+9,629+9,791+16,734=96,445 genes from AraTha, ChlRei, ChrZof, CocSub, Chlore, and MonNeg, with each gene belonging to exactly one final family), with 30,838 and 10,490 involving single vs. multiple organisms, respectively. Of the 10,490, 5,012 have ≤1 gene (primary+additional) per organism and 7,904 have ≤2 genes. The largest families are of various histone proteins.

TABLE S4 % of reference genes Same, except multi-orgo. family Same, except multi- Reference that belong to multi- restricted to having ≤ 2 genes organism family has ≤ 1 genome: organism families: (primary + add'l) per organism: gene per organism: AraTha ~60% ~18% ~7% ChlRei 64% 37% 21% Chlore 83% 52% 29% ChrZof 73% 47% 27% CocSub 77% 50% 26% MonNeg 63% 39% 20%

Details of Main Text FIG. 2

Phylogram.

The 813 protein-coding gene families (called across the six organisms of main text Table 1) that have no additional genes and exactly one primary gene in each of the six organisms were identified. Because of the highly fragmentary nature of the MonNeg assembly (and the possibility of artificially truncated gene coding sequences), an additional condition that the shortest protein across the six organisms is ≥85% of the length of the longest protein was also imposed, resulting in 75 families with an average of ≈27K amino acids per organism. Multiple alignments and phylogram estimation were by the ETE Toolkit sptree_fasttree_all/standard_fasttree pipelines (56, 57). Alternatively, if MonNeg is ignored, there are 1,253 families before the similar length requirement, and if this requirement is loosened from 85% to 50%, 978 families with an average of ≈497 K amino acids per organism proceed to the same ETE pipelines, and the resulting phylogram is very similar to that shown with just a slightly higher average rate of amino acid changes but similar proportions; this phylogram was stable when the 978 families were randomly partitioned into six groups of 163 families each. An analysis based on 16S/18S rRNA nucleotide sequences extracted from NCBI also produces a similar phylogram (but with a much lower average rate of nucleotide change). The topology of all these trees is in agreement with Leliaert, et al. (58).

Proportional Venn Diagram.

The protein-coding gene families containing the 15,274 genes of ChrZof were partitioned into eight classes based on the subset of MonNeg, ChlRei, and AraTha that have at least one gene (primary or additional) in the family. The number of ChrZof genes in each class is shown as a proportional Venn diagram using eulerAPE (59)-determined ellipses.

Scatter Plot Showing Scrambled Syntenic Blocks.

This is similar to FIG. 2 for CocSub vs. Chlore in Blanc, et al. (51), but with a finer scheme for generating statistical enrichment shading as well as permutation of genome assembly sequences to emphasize enrichments. To identify statistically enriched regions, each assembly sequence is partitioned into as equal-sized pieces as possible with each piece being ≈1 Mbp (small sequences are taken whole); this induces a 2-D partitioning of the plotted area, and the number of observed gene pairs (red plus green dots) in each 2-D bin is noted. Randomized versions of the plot are then generated: for each version, the identities of all genes are shuffled in each genome and new numbers of points in each 2-D bin tallied; the p-value for a 2-D bin is taken as the fraction of times the random tally is larger than the observed tally over 100,000 randomizations. These p-values are used to shade the background of 2-D bins from white (p-values above 0.01) to increasingly orange on a logarithmic scale to deepest orange for p-values near 0.00001.

The plotted order of genome assembly sequences along each axis is determined as follows. Reordering is only performed among those sequences (“large”) in an assembly ≥0.5 Mbp long. First, consider 2-D bins with p-values at or below 0.01, and form a directed graph with arcs from x-axis large sequences to y-axis large sequences with arc weights given by the total number of red plus green dots in considered 2-D bins that land in the pair of sequences, deleting arcs of weight zero. Using the Centrality method of FindGraphCommunities [ ] in Mathematica, partition the sequences into an ordered list of clusters. Start by considering in turn those clusters that involve both genomes: find a maximal-weight matching for the subgraph of the current cluster, place ordered pairs (x-axis assembly original sequence number, y-axis assembly original sequence number) for the matching is ascending lexicographic order, and take these as the next sequences in the reordering for both genomes; if the matching does not involve all sequences in the cluster, add the leftover sequences by ascending original assembly order. Finally, after all clusters involving both genomes are processed, in each genome add in all sequences not yet included in ascending original assembly order.

Gene Prediction and Functional Annotation

Ab initio gene models were constructed with AUGUSTUS 3.0.3 using default parameters except where noted as follows. PASA 2.0.2 (50) was used to extract a training set of 6,576 genes from the assembled transcriptome. Prediction hints for AUGUSTUS were created by aligning the transcriptome to the genome with BLAT 35. Functional annotations were generated from protein translations of predicted gene models. For example, BLAST2GO 6.0 (60) was used to associate Gene Ontology (GO, 61) terms as well as brief textual descriptions to genes. To generate protein domain/family annotations, protein translations were scanned against PfamA release 29 with HMMER 3.1b2 (62). Additional GO associations were derived using the Pfam2GO translation table from EMBL-EBI (63). All functional enrichment analyses were based on hypergeometric statistical tests using the annotations of the entire genome as background.

Astaxanthin-Deficient Mutants

A non-targeted forward genetics screen generated astaxanthin-deficient mutants. Cells were grown to log phase (2-5×10⁶ cells/mL), subjected to ultraviolet radiation (80,000 μjoules), and plated onto selection media (proteose media with 28 mM glucose). The selection media enhances the production of astaxanthin, which causes the cells to become pink; therefore green colonies were selected as astaxanthin candidate mutants. The lack of astaxanthin production was confirmed by HPLC pigment analysis. To analyze pigments, cells were scraped from plates and homogenized with acetone and lysing matrix D for 2×60 s with the FastPrep-24 (6.5 m s⁻¹, MP Biomedical). The cell debris was pelleted by centrifugation (20,000 g for 3 min) and the supernatant was removed. To ensure complete extraction, another aliquot of acetone was added to the cell debris pellet and the extraction process was repeated; pigments were determined by HPLC as previously described (64). To sequence the β-carotene ketolase gene from C. zofingiensis wild type and astaxanthin mutants, a series of synthetic primers (Table S3) were used to amplify overlapping fragments of genomic DNA. Sequences were assembled using Lasergene MegAlign (DNASTAR) and putative point mutations were identified.

Liquid cultures of wild type and astaxanthin mutants were grown until log phase under medium light (100 μmol photons m⁻²s⁻¹) and then high light treatment cultures were moved to 400-450 μmol photons m⁻²s⁻¹ for 10 days. Replicates (N=3 or 4) were harvested by centrifugation and the cell pellet was frozen in liquid nitrogen. Pigment determination was conducted as described above. Pigment concentrations were tested for assumptions of normality and homoscedasticity, and data were log-transformed accordingly prior to analyses. ANOVA was used to test the effects of high light. For all significant factors in the ANOVA tests, post-hoc Tukey-Kramer HSD pairwise comparisons were used to test which groups were significantly different. α-carotene concentrations were not normally distributed and the Kruskal-Wallis non-parametric test was used instead to evaluate statistical differences. Statistical differences were reported significant at the α=0.05 level.

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Table of Illustrative Sequences: Amino Acid Sequences: SEQ ID NO: 1; amino acid sequence Cz02g29020 without the starting methionine EAALTTAVDAAAALRNNIGFLQRSQIAIVAAFLSAIYLKLVVSRIAPGWKSLALVAPLLALNLWLPLLFH MREEVVSRTCVVFLISWLGSFKAIGMCLNRGPLAADWSVAQMIFLYVAPVLPRQDAGGPVKAGRLQDSAG TWGTLAASFVINTTILGVVSYLLVVMDMPKVVKIYIYALGLYGFVSFIMEGPAAIIVGLLRIELVPPFDK PWLATSLADFWGRRWNNSTSLLLRFLVYDPIIEGRLVKKAKQEDPQQPAPSKQVSNSMRLTAMLATFALS GLIHEAILAYVNRPYYPGPWFMFFFIQGPLLAVEMRVHKHLRATHKQLPFTAAWTLTTACLLLTAYMFFF PPIEDWTDLAPRIAQAVTSNFQAVLGPLQGLGRQYWAVAAGLVQQSAI* SEQ ID NO: 2; amino acid sequence Cz04g21110 without the starting methionine PVPVSTTHPNGKGMRAANLTELPTEGPFSRSLKQKEDSANTGADAIALQTNGTADASLDANAPSPLPEPG PSVLVHHLDFTYPGLDGRPVPGQPPLITDMCLTLQPGSRCLLIGANGAGKTTLLKILGGKHMVPKDAVHI LGAPPFHDTHLTTSGHLSYIGGNWTRDIAFAGLSIPLTGDFPASRMIDAIPEVDPARKERLIKVLDIDPN WRMHTVSDGQRRRVQICVGLLRPFKVLLLDEITVDLDVLGRADLMHFLADECATRGASIIYATHIFDGLE FWPTHVAYVARGRLQMVKSAEEISELKQGRLLELVTSLLREEREAVKAAGVSKPLEYDPSLEGQVTNFSY AFNNGWVPGTLSTSLAKSTNAVMRN* SEQ ID NO: 3; amino acid sequence Cz05g17060 without the starting methionine LQRIVGGLQCYARQQAVPSVQCPENAYLYYWIIGLSLAPLLQSLAENSMNFELSVIGTRMRNGLMAAIYR KCLRLSNSAIQSESTGKVVTLMSNDAQKVQDAMMAIHTIWGAPALIVVITILLYQQVGWATFVGLGIMLI YAPMTGKVSRKLVVLRRSIMQWTDRRVGLMNEVINGMQMIKFYAWENSFKRQILHARDNEARILKTVVLW QSFFAMLLFSGPVAVAVFCFGSWALAGYGLTAASAYTALSLFSLLRMPLVMLPMMITMIINALVALNRIS AFLLRGEXQTTSQPGHDGNSSKEKQETVQETVQETVQETVEPGVVRVTEGTFSWDSAGDQAALRDVNFTA APSSLTMVVGSVGCGKSSLLSALIGQMEKQGGEVQLGGRVAYVAQTAWIINDMVQENILLGEAFDADRYR MAVEISQLVPDLELLPNGDRTEIGDRGVTLSGGQKQRVSIARAVYSDADVYLFDDPLSAVDSHVGRALFE KCIRGVLRNKTVILVTNALQYLPSADNILWMEGGAVKAQGSYSQLVAAGMNVAELVHVEHEAEQTHQRDE DPSRQAGTGVVAEEADLPDDHDSTGTSGTDSAYSDDIAKATIVSHDDADGDKAARHVKGSYIPEGKPSKS ITLRKIDTVDNRNLTGIEARETGAVSGKVLKSYIIAGGGFIIIAFVALLFAAEQGARVFTDTWVGLWFGD AFRQNVWFYVGIYAALGILYSFLTFLRALRFNYSAVNAALSLHNQLLNHILRLPKSFFDTNPAGRILNRF SRDTEIMDSTVATSALMFCNCMATFIAILIVISVATKWFAVAIIPITITYMLLQRYYIPSARELQRIESV SRSPIYSKFSEALAGVPTIRAYRKERYFTVTSDKLMQENAYAYISQRSAASWLAMRLDVVGVLILTLTGV LCIQGTISPGLAGLCLVYALDLTRYLKMGTAMASKTESDFNSVERIIQYLEPAPEADADTPADVLATLPA NWPAAGSISVRDVCMRYRPGLPLVLKGVSFDIAPGEKVGLVGRTGSGKSSLFLALFRMAEPESGSILIDG VDIRTLGLHTLRAAMSVIPQDPFMFSGTVRHNLDPFDEHPDSELWRVLEAVGLKTVISVLEAKLEAPVVD NGANFSQGQRQLFCMARAMLRRSRILMLDEATASVDPETDSLIQAAIRSAFQECTLLTIAHRLNTIMDSD RVLVLDGGVVAENDQPHHLLQNNNGLFTQMVDQTGKQSSTYLKQVARSATTSRQTARRRMAVAHHDTAEI TDMAGLRSESTVGVVPQLSPTMDPLGPAVEAAPARLTTEVSQLRRNIDAGDVALTSPTVAL* SEQ ID NO: 4; amino acid sequence Cz08g16130 without the starting methionine KADADVCIKDETVVDMKRLELDNGMPGVVSGGSHHLPAKRGSSFGPSGMTISVKDLTFSVKSNIEKGKTV HLLKNVTGFFEPNKMSALMGPSGSGKTTLLDILAGRKTSGKTEGTILFAGNKPTRQFLRRYTGYVEQFDT LLPTLTVEEMLMYTAELKRPISEPLSEKKAAVDELIDKLALESCRKVPIGSSMSKGISGGQAKRTNIGIA LITNPRVLFLDEPTSGLDSFTSNEVMTVVKALVSDGVTIVATIHSPTAYAFNLFDKLMMLVKGRVVYFGA QGKPALEYVRTQCPQIKEQSSGYGSDAEWLVDLFTEADRMGKGGEFADAYDVSQLKKDNDYIVDSLCAQK HVLPAHVQQELSVKTETVTPWWWGIKTLIKYRTTHNYRDAAFLGPRIGDKLLIGLLIMTLYLGIGDDFAP DNVINIAAVNFMFVTMPAFGAAAYVPAIVLERNLFCRERNDGVYRVITYLMAKMLDELMIAAVASCVIAA IAFYGIQLQGEFVLFWLVYYIVLCTGIVLAYFVAALSPNMDVANAALPTYVTSLLFFGGFLFTFDKMPVW WKWYSYIDVIRYAWTAIMVNQFEGRDAQMFSGQTVLQYYGIEGQNKWANLGYTACFFFFFTFCAWVTLSV KKYQRR* SEQ ID NO: 5; amino acid sequence Cz09g27180 without the starting methionine QLQFTNQVGQARLRAAVPRPGHKHAGVRARPFQAAKPGCRPPTILASSAALSSIDYTYSSSVSDAKSRPF PAVAPLPSPPRTADLGNVLPYLAKLAVGERQLLWRFGVALICMVTSKLAGLAGPVLLREAVNAVGEQATA SLRPAVHAVVCYGLCGVLGTLAKELQHPTFAPVSQAVARRVAYHTFAHVLDLDIKFHLERRTGRLSRILE RGTRSVQMLYRAVLFTFIPTALEFAFVIGLLGTQFSSTVAGLVAVTFVAYVAWTLAMTQSAVEVRKQVNT LDNLTTSKAVDALLNAETVTLFNNQALEVQQYDHYLRGFQRAAIQTERLSALLNAGQSAILTIGLMLVLI AALVSAPATRPVTAGDLVLLQGLLLQLWSPLQFLGWFYRELRQSLVDMEEFFEILQTQSQLPDGHLSLPN TPPCMVRNIAAQTSNSTSRNGNSSQRLGSNTVSSTGTSPHHSHSQQQQQPDDPTASYHEIPYDAACISGF GLEVELKDVHFGYHPDRQVLRGVTLRIPPGQSVAIVGSSGSGKSTILKLVTRLYDVTTGSVEVNGVDIKD LTRDSLRAAVAVVPQDTVLFNDTILQNIRYGRPESTDDEVIRAAHLAHLHDAVVKMPEGYKTVVGERGLK LSGGEKQRVAIARAFLRAPRLLICDEATSALDSATEASIMNSLNELAQGRTSLFVAHRLSTIRNCDRIVV LSAGVVVEEGTHDQLMSRGAVYRDMWEMQAKEASRGNGVAEGTTSDSEDGERTMEPLPAALTAKSLN* SEQ ID NO: 6; amino acid sequence Cz10g28330 without the starting methionine MHTSSSTLYPLGIISFLVIVVPLVMHVLSSRRLDLDLLPSPPAVLLLGHIQLALHVKDMHLQFLRWHNRF GKLLRIRVLQQDMVLIADPALASEVLTLGPNYCARRPADYATFNVIHGLSARPSILTHQDEAWWKAVRRA IAPAYTPAATRELHSLMVSTMARVCERINANLHSAANNGNSTSTSSSDSTGSPAGIRMDEEVLVAVLEIL LHGSLRLPPQAMSSSDVRRTAQVAPKLVTIANSFVSLPGKQWVYTTLPFICKEARVMHQGRTHMTNITRQ IYNHISAKYTVNGVPSAKSDVGGSEKSDTSLASCLMRLAHPQHTPGQTFTQDDIMAEIAINIIGQGSVPW TVSWALFQLTQRPDVESRLLSELQGLGLPCDGDLAAACDAISHPECLRDTPYLTAVINEVMRMYPAGVSA APRLTEQPIKLGGYRIPAGVMVFPNLFTIMNYRGNWQQPDQFYPDRWLQPNSSVDPSSGAPRFIPFSIGP KICIAQHLAIMQCKIMLTLLVSCLKFQLAPTMGGIEGVMKRVEATLELRVQGGLWFTAEIRRSNNVK* SEQ ID NO: 7; amino acid sequence Cz11g14160 without the starting methionine DGLAIAMGCAAAVLVCYTVLPFLCSLALAIWRFHTSKIPGPPAKDFILGHAAAHLNDKAPFIFKRWAEKY GKLYKVRMADNFAVVLTDPEVIQSIARKIQKPTKSYSSLELGTFPKDHNILTAPDGPHWKAVRQGITPAF SVANLKQVFPWLCHVTKVAAAKLQDTGPSTSFDISDIAKRITSDVIGQLLYAEDLGAIAYRPSEYLELFQ IGITAAHKSLGRPLRLYAIWDPEVRRQNRAINRLDQ* DNA sequences: SEQ ID NO: 8, gene sequence (starting from ATG) Cz02g29020 ATGGAAGCAGCACTGACCACTGCCGTAGATGCAGCGGCAGCGTTACGAAACAACATAGGCTTCTTGCAAA GAAGCCAGATAGCAATCGTAGCAGCGTTCCTGTCAGCAATATACCTCAAACTGGTTGTGTCAAGGATAGC ACCTGGCTGGAAAAGCTTAGCACTTGTGGCTCCGCTGCTTGCATTGAACCTGTGGCTCCCGCTACTCTTT CATATGAGAGAGGAGGTCGTCAGCAGGACTTGCGTGGTATTCCTAATATCTTGGCTCGGCAGCTTCAAGG TAGCAGTAAGCATAATGATGTAGCTGTTAACGCAGCAAGACTGGCCTGCATGCACAATTGTGTGGTCAGG CACACCGACCATAGCCAGCAGCCACAGCACAACCACATCAGCTTGTATGTATGTATGTTCAATGAATGTA TGCATGCATCACATCTAGAGCGCAACGTCCTCAAGTACCTTAAGTTAGCTCATGCTCAGAATCGTGACAT GTCTGGTGCTGCAACTTGCCAGCAGTTGCACCAATGATGATGATGATAATGGTCATACCTGGTTGGCATT ATTGTGAAGCCATCACACATGCATGCTATCATTGCAGGCAATTGGTATGTGCCTCAATAGGGGGCCACTG GCTGCTGATTGGAGTGTAGCACAGATGATCTTCCTGTATGTTGCACCAGTACTACCCCGACAAGGTACAT GGCATGGCAGACACAGGTCCTTAGTTTGTGGTGTTGCTTTTCTCGTTGCTTACGAAGCAGTATGCAGGAA CTTAAGTTATTGACATGCAACACCCAGCAACAACAGGGCAAGAAGTAGGATGCTTGTGAATGCATGTCGG CATTTTCCAATGCCTCTGTTCTGAACTGTGGCGGATGCGGACCGTGCATCCACCCTGATGAATTTCAGCC CTCTGTCTCTCACTCTGATTGGCATATACTGACACATGTGTTGTGCTCCTGTGTCATATGCAGATGCTGG CGGCCCTGTCAAAGCAGGCCGGCTGCAGGACAGTGCTGGCACCTGGGGAACTCTTGCGGCATCATTTGTG ATCAACACCAGTAAGTAGGCGCCACAAGCAGTACATGCCAGACTGAGAGTTGAGGCTGCCCTCTTTACAC AGCATTCCTGGCTCATGGTGCTGTGGCTCATTGCAACACAACTGCATCATATGACATGCTGCTTCTACAC ATTGTCCTTACACAATGGCGCCAGTTGTTGTTGTGTGCTCCACCGCCCATCAGTTGTGATGAGCTGAATA GCGACTGACAATAACCACTTGCTTGGTGCTGCTTGCAGCAATTCTTGGTGTTGTTTCTTACCTACTGGTG GTGATGGACATGCCAAAAGTAGTCAAGATATACATCTACGGTGCGCAAGGCAGCCCACATAGTTGCTGTA ATCCACTACGCATCATAACTGTCTATGTGTGTGTCATAGGAAACTTGCTGCTGCGTTATGACCTGTAGCT GTCATACATTGGCAGTAGTTGTTACCCTCTTGTGTGTGCTTGAGCGTGGTCATAAGTCATGGTTTGCATG GCCAGATGACATCAGCTTCTGCCCAGGAATACTGCATCTGCCTGCCATCCGCTACAACATTGCTAAAGTT GATACTGAACCTGTCTGTGATCCTCTACCATCCTCTACCTTGCAGCATTGGGTTTATATGGCTTTGTCTC GTTTATTATGGAGGGTCCAGCAGCCATCATTGTGGGCTTGCTGAGGATCGAACTTGTGCCTCCCTTTGAT AAACCATGGCTGGTAAGTGGCTGACATAGACCCAAAGCAAAGCAGTAAGGCCATGAGGCCCTTAAGGCAT GGAGGTGTGCACAGTGGACAGTTTTGCACACTTGTCTGAAACAGTTGAGCTTCACTATGGTCCTTGTGAA GGAGCTGAGTGGCTTGTGCATGTTGCGTAAAGGTAGCCACTGTGTCTACTAACACTGACCTTCATGTTAA AATACATACGTGTGTTGTGGTGATGGTGACGTTGATGCTGCCGCTGCGGTGCTGATATTGTTCTTTTTCC TGTTCCACGCAGGCGACTAGTTTAGCTGACTTCTGGGGTCGGCGTTGGAACAACTCCACTAGTTTGTTAC TACGGTTCCTGGTGTATGATCCCATCATTGAAGGCAGGTTGGTGAAGAAGGCCAAACAAGAGGATCCACA GCAACCTGCTCCAAGCAAGCAAGTCAGCAACAGCATGAGGCTGACTGCCATGCTTGCTACGTTTGCATTG AGTGGGCTGATCCATGAAGCCATCTTGGCGTATGTGAATCGGCCATACTACCCAGGACCCTGGTTCATGT TCTTCTTCATTCAGGTGAGCCTGGTGCTGAATGATTGATAGTAGATGCATAATCAATGATTGAGGCACAC TGATCTTAGGCCGTGGTCATCGTTGCAATACAACATTACTGCATGTCGTTATGTGGCATACTTCTTGTTG TTGGCAGTGCAACACCTCAGGGGTGGTCAACTGGCCTTGGGCATTACAGCCTTGCAGACATGGGTATGAC AATCACACGGTTGATGGTAAATTGTTGTCTTGTAACCACGCATGACTCCTCATTTGGCAAGTGCCCATCT GGGCACCACTGTTATACCGCTTGGTTTGTGCAACTGTTGTCTTGGCTGATTTGTCGACATGTTGATCATT GCAGGGGCCCTTGTTGGCAGTGGAGATGCGGGTACACAAGCATCTGAGGGCAACACACAAGCAGCTGCCA TTCACTGCTGCATGGACCTTGACAACAGCATGTCTGCTGTTAACAGCTTACATGTTTTTCTTCCCTCCCA TCGAAGACTGGACAGACCTTGCACCAAGAATAGCTCAAGCAGTCACCAGCAACTTCCAAGCAGTGTTAGG CCCGCTTCAGGGGCTGGGGCGCCAGTACTGGGCTGTAGCAGCTGGGTTGGTGCAACAGTCAGCCATTTGA SEQ ID NO: 9, gene sequence (starting from ATG) Cz04g21110 ATGCCGGTACCTGTGTCCACAACCCACCCAAATGGCAAGGGAATGCGCGCAGCCAACCTCACGGAGTTAC CCACAGAGGGGCCTTTTTCACGATCGCTGAAGCAGAAAGAGGATAGTGCCAACACAGGAGCAGATGCAAT AGCATTGCAAACAAACGGCACTGCTGACGCCTCGCTTGATGCCAATGCACCATCACCATTGCCAGAGCCG GGACCATCAGTGCTGGTTCATCATCTAGACTTCACATACCCAGGGTTAGGTGGGTGCCTGCCACATTGTC ATCATTGGCAACAGGCAACCGAATGTATCTTTCACAGTCACATACATACTGCAAGGTGTTTATGAGTCCC ACAGACATCAGACGTGCTGGTGCATTGTACCCTGTGCCCAATTCATCAAAGTGCATGTGCACTTTAGCAA GAGGGATGGGTATTTTCACAGATGCTTTGTACACTCATTGCATGCTCTGTGTTTGCAGATGGCCGCCCTG TGCCTGGTCAACCTCCTCTGATAACCGACATGTGCCTGACGCTACAGCCGGGCAGCAGGTGCTTGCTGAT TGGCGCTAACGGTGCAGGCAAAACAACGCTGCTCAAAATACTGGGTGGCAAGCATATGGTACCCAAGGAT GCTGTTCATATACTGGGTGCACCACCCTTCCATGACACACATCTCACCACATCAGGGCACCTGTCATACA TTGGTGGTAATTGGACTCGTGATATTGCATTCGCAGGATTAAGCATACCGCTAACGGTGAGCACAAATCC AGATAACTGTTTTGTATTTGTGTATGTTATCACCAAAGCTGAACTGACACCACAGTTGCTTAAACAGCAG TGCATGGTGCCTGGTTGCCCTACTTTGATGCCTGGGCCATAGTACTTCCATATCCCATCTTCTCACTGCT GCAAAGAAGCTTGCTTTTGTTCCTTTATGGGTTCAGAGCAAAGCAGCTTCTGACAACCATTGTTTTAACC ATGTGTTTGTCAGTTTATGATTTTGGGTTTGTAGATAATGGGTAAACCTTTTTTGTTTTTGCTGACCCAC TACATGGTGGCTGCAGGGTGACTTCCCTGCTTCACGGATGATTGATGCCATCCCAGAGGTTGACCCCGCA CGTAAAGAACGCCTGATCAAGGTGCTGGACATTGATCCAAACTGGCGCATGCATACGGTGTCAGATGGTC AAAGAAGGCGTGTGCAGATCTGTGTTGGGTTGTTGCGCCCATTCAAGGTGGGGCTTCTGATGGTGACAAT CTTTATGTCAGCAGCTACCACCAAGTAAATTGCCAAGCTGTTGGTTGCGTATCAAACAGGCTGGGCAGGA CTTCTATGCCTACCCCCTTGCTTGCTGCGCAGCTGGGTTGATATGAAGTGCAAGTGTGCATGCATGGTGT ACCGAACAGTTCTTGGATGTGGCAGCCAACGCTGAACGGGTATATACTTGCATGGCTGCCTGATGCAGGT GCTGCTTCTGGATGAGATTACCGTGGACCTGGATGTGCTTGGTAGGGCTGATCTGATGCATTTCCTGGCT GATGAATGCGCCACCAGAGGAGCTAGCATCATATATGCCACACACATATTTGATGGTCTGGAGTTTTGGC CCACACATGTAGCATATGTTGCAAGAGGGCGGCTGCAGATGGTCAAGTCAGCTGAGGAGATATCTGAGCT CAAGCAGGGCAGGTTGTTAGAGCTTGTCACCAGCCTGTTGAGGGAGGAACGCGAGGCAGTGAAAGCTGCT GGCGTCAGTAAACCACTGGAGTATGATCCCAGTCTAGAAGGACAGGTTACAAACTTCTCATATGCCTTCA ATAATGGTTGGGTTCCTGGAACGTTGAGCACCTCCTTGGCCAAGAGTACTAATGCTGTGATGCGTAATTG A SEQ ID NO: 10, gene sequence (starting from ATG) Cz05g17060 ATGCTTCAGAGGATAGTAGGCGGGCTTCAATGCTACGCAAGGCAGCAGGCTGTACCGTCCGTGCAGTGTC CTGAGAACGCCTATTTGTACTAGTAAGTGCAGAGGGAGACTTATTCTCGCTGCAGCTGCATGCATTGGCT AGCAGCACTGTCGCATGCATGGTGCATGTATGGTGTGCTCATGCGTGTATGACGCACATGCATGAGCACA TTCCTTACGGGGAGCCCATTTCCATGTGTAGGCTGTCAATGTTGTGTCATGCTCTGGTTACATGATTGCT GATGCTAGGGTATCTGTCAGCTGTGCCATGCATGCTTATGCTAGCATCACTGCTGCTGTAACTATTACCA TACATGGTTGCATTACATCTCTTGCTGTGTTTGTGCAGTTGGATCATAGGTCTGTCGTTAGCACCTTTAC TACAGAGCTTAGCAGAAAACTCCATGAATTTTGAGCTCAGCGTCATCGGCACACGCATGCGCAATGGACT GATGGCAGCCATATACCGCAAGTGTCTGCGGTTGTCCAACTCAGCCATACAGAGTGAGAGCACCGGCAAG GTGGTCACACTTATGAGTAATGATGCTCAGAAGGTTCAAGATGCCATGATGGCCATCCACACCATCTGGG GTGCACCTGCCCTCATTGTGGTCATTACCATACTGTTGTACCAACAAGTGGGATGGGCAACCTTTGTGGG ACTAGGAATCATGCTTATCTATGCACCCATGACAGGTGAGGGCACCGGGAATTAGTGCTGCCTGCTTCAT GTTAAGGTACCCCAGATGTATCCTACATGTGCCATGGAATGCGGTCTGGCAGTAACGTCTGGCAGTAACC CAAGAGGTTATCGTTACCATACGCCCAAACATGCATGTGCAAACCGTGTTGATGTCTGTGTTGCAGGCAA AGTGTCACGGAAGCTGGTGGTACTGCGTCGCAGCATCATGCAGTGGACAGATCGCCGCGTGGGCCTCATG AATGAGGTCATCAATGGCATGCAAATGATCAAGTTCTATGCCTGGGAGAACTCCTTCAAGAGGCAGATCC TGCATGCCAGAGACAATGAAGCCAGAATCCTGAAGACTGTGGTGTTGTGGCAGTCATTTTTTGCAATGCT CCTGTTCAGCGGCCCTGTGGCTGTAGCAGTATTCTGTTTTGGTTCCTGGGCATTAGCTGGCTATGGCCTG ACAGCAGCGTCAGCCTACACAGCACTGTCACTGTTCAGTCTGTTGCGAATGCCTCTTGTCATGTTGCCTA TGATGATTACCATGATCATTAATGCACTGGTGGCCCTGAACCGCATCAGCGCGTTCTTGCTGCGTGGCGA GATACAGACCACCAGCCAGCCAGGTCATGATGGCAACAGCAGTAAAGAGAAACAGGAGACTGTTCAGGAG ACTGTTCAGGAGACTGTTCAGGAGACTGTTGAACCTGGAGTGGTGCGGGTACGTTGTGTAGTGCTGCTAT AATCAGCTGCTTAATTCTGGAGTGGCAATGTGAACTCACTGATATCAGTCTGTTGATTGTGGTCTGTGAA CTTTGAACAATGTTGTGGTGTGGCTGGCATCATCCAAGGTACCTGTTTTACAGCTTTGCAGCCACATCCA CAGCCACAATGTGAGTTTGTTGTATGTTGCAGTGTTCCGTGGATGTGTGTGTGGACCAAAGTAGTCACAC ATGTCTAAGTCAGTCTCAAACACTGCTGTCGTCAGGTGACTGAGGGGACCTTCAGTTGGGACTCTGCTGG CGATCAGGCAGCCCTGCGTGATGTCAACTTCACTGCTGCCCCTAGTTCATTGACCATGGTGGTGGGCAGT GTGGGGTGTGGCAAGAGCAGTCTGCTGTCAGCATTGATTGGCCAGATGGAGAAGCAAGGAGGAGAGGTGC AGCTTGGAGGCAGGGTTGCCTATGTTGCACAAACGGCATGGATCATCAACGACATGGTGCAGGTGGGTGT GGCTGGCCACCTTGTGCACCTTCTTTGATTCAGCAAAGCAAAGCAATGTTACAGCCTTATCCCATGGTCC ACTCTCTTGGTTACCTAGTAATGGTATGCGCGTATGGCAATACAAGTGCCATACAAAGGGCCCCACATAA TCTATGTTGGTCATGTGTTCGTTCTGATCAGCTTGAGCTGTTGTGGTTGCTAGGAGAACATCCTTTTGGG TGAAGCATTCGATGCTGATCGGTACCGTATGGCAGTGGAGATCTCTCAGCTAGTGCCTGACCTAGAGCTA CTGCCAAATGGTGATCGCACAGAGATTGGCGACCGCGGCGTCACCCTGTCCGGGGGCCAGAAGCAGCGTG TGTCGATTGCAAGGGCTGTATACAGTGATGCTGATGTGTACCTGTTTGATGATCCTCTCAGTGCTGTTGA TTCCCATGTGGGTCGAGCACTGTTTGAGAAGTGCATACGGGGTGTCCTGCGCAACAAGACAGTCATCCTG GTGACTAATGCCCTCCAATATCTGCCCTCTGCGGATAATATTTTGTGGATGGAAGGTGGTGCTGTGAAGG CTCAGGGCAGCTACAGCCAATTGGTGGCCGCTGGCATGAATGTGGCTGAGCTGGTACATGTTGAGCATGA AGCTGAACAGACACATCAGCGGGATGAGGACCCCAGCAGGCAGGCAGGCACGGGTGTAGTAGCAGAGGAG GCTGACCTTCCTGATGATCATGACAGTACAGGCACGTCTGGCACTGACTCAGCTTACAGTGATGACATTG CCAAAGCTACCATCGTTTCTCATGATGATGCTGATGGTGATAAAGCAGCCAGGCATGTGAAGGGGTCCTA CATCCCAGAGGGAAAGCCATCAAAGTCCATCACGCTAAGGAAGATAGATACTGTAGACAACAGGAATCTG ACAGGTATTGAGGCTCGTGAGACGGGCGCTGTGAGTGGGAAGGTGCTCAAGTCATACATCATCGCTGGCG GCGGCTTCATCATTATAGCCTTCGTTGCGCTATTGTTTGCTGCTGAGCAGGGTGCCCGTGTGTTCACGGA CACTTGGGTTGGGCTCTGGTTTGGTGATGCGTTCAGGCAGAATGTGTGGTTCTATGTGGGTATCTATGCA GCACTGGGAATACTGTACAGTTTCCTCACTTTCCTCAGGTAGGTAAAAGTCGATCTGTTGGGCAGTATTC ACATGCTGTCATTTGTTGCTTTGTATTTTGACTTTGAACTTGAGCGTGGTTCACTGCTGGTACTGTTGAC TGCATAAAAGGTGCTGATGTATGTTTCTTCTGGCGTGCATGGCCCATTCAGTGTCTTGTTGGTGTTGCTG CAGTGTCTGGTCAGTCTTTGCTGTGTGAAAGACCATGCATATGTCGGCAGCTGCCTCCAAGTGTCTTCCC TTGGAACCTTTGTGTGTAGGGCACTTCGTTTCAACTACTCAGCCGTCAATGCAGCCCTGTCTCTCCACAA CCAGCTGTTGAATCACATCCTGCGCCTGCCCAAATCATTCTTTGACACCAACCCAGCTGGCAGAATTCTC AACAGGTTCAGCAGAGACACTGAAATCATGGACAGCACTGTTGCTACATCAGCGCTCATGTTCTGCAACT GCATGGCGACATTCATCGCCATCCTGATTGTGATCAGTGTTGCAACGAAATGGTTTGCTGTAGCCATCAT CCCTATCACCATCACATACATGCTACTGCAGGTGAGCAGCGTGTCGCAAATTGCAGTTTGTTTATGTGCA TGTCACTGTGTGCTTGTTGGCATGCAGGTACAGTGTATTGTGTTTGCAGTGCCGTGCACTTGCAGTGAGA CAATCATATGTCTGCAAAACAAGCTGTAGCGCTGGAATTGTTTTCTAACTCAGCCGTTCTTGTTGTTGTC CTGCAGCGGTATTACATCCCATCCGCTCGTGAGCTGCAGCGCATTGAAAGTGTGTCCCGCAGTCCCATCT ACAGCAAATTCAGTGAGGCGCTGGCAGGCGTGCCCACCATCAGGGCTTACAGGAAGGAGCGGTACTTTAC AGTCACCAGTGATAAGCTGATGCAGGAGAACGCATACGCTTACATCAGTCAGAGGTCTGCAGCTTCATGG CTGGCCATGAGACTTGATGTTGTGGGTGTGCTCATCCTGACATTGACAGGTGGGTGGCAGGAGTGCCTGC CACATGCTAGCTTCACTTGTAGCTTGCTTGCTGGCAGTTGTTACAGTGTGCATTGCTGCTGCAGCTTGGG TTCTGATGACTGTTGCACTATGTTTGCTGGCGGCACCCTCTACCTGTATGCTAGTTTTTGTTTGAGATGG TTGCTGTTGGTATATGGATACACCAGCAGTCAGGATGAACTGCAGAGCTGTTATCCTCCGGCGTTGGTTG TAAGTGTGCTACATATGACGTGTTTTGCTGACAAAGACGATTGCTGTTGTTCCGCCAGGTGTGTTGTGCA TTCAAGGCACCATCAGCCCCGGCCTAGCAGGTCTGTGCCTGGTGTATGCACTGGACCTGACACGCTACCT GAAGATGGGCACGGCAATGGCATCCAAGACAGAGTCTGATTTCAACAGTGTGGAGCGAATCATTCAGTAC CTGGAGCCTGCACCTGAAGCTGATGCTGACACACCCGCAGATGTGCTGGCCACACTGCCAGCCAACTGGC CTGCTGCTGGCAGCATCAGCGTGAGGGATGTGTGCATGCGCTACCGCCCTGGGCTACCTCTTGTGTTGAA GGGCGTCAGTTTTGACATTGCGCCTGGTGAGAAGGTGGGGCTGGTGGGCAGGACAGGCAGTGGCAAGAGC TCACTGTTCCTAGCACTGTTCAGGTGAGCATCATAGAGGGGACGACACCGGCAGCAGCAGCAGAGGCATG GCACAAACTGGGAGTTATGTGATTGCTTGTTGCTATGTATCATCGCATACATTTCCAACAGACAACCGAT GTTGTGTGCCGGACTGTGTTGGTAACGTTTGCTTAAGTGCCACAGGTCCTTCTACTTGCAACTTGTACAC TCCTGGGACTGTTGTGTTTTTAACCACACCATAGCCCACAGTGTACTGCGTGTTGTTGCATCTTCGCCTC ACTTGCTGGTTCTTGCATTGTGTAGGATGGCCGAACCAGAGTCTGGCAGTATTCTGATCGATGGTGTTGA CATCCGTACCCTTGGCCTGCACACACTGCGTGCTGCTATGTCTGTCATCCCTCAAGACCCCTTCATGTTC AGCGGTACTGTGCGGCACAACCTGGATCCATTTGACGAACATCCTGACAGTGAGTTGTGGCGCGTGCTAG AAGCGGTTGGGCTGAAGACAGTCATATCTGTACTGGAGGCAAAACTAGAAGCTCCTGTGGTGGACAATGG TGCCAACTTTTCACAGGTAAATTGTAGCTGCTGATTGACAAGGCATTGACAGGGTTTGAAATCCATGCTT AGACTGATGTGCTAACATGCATTCACGTGTGATTACCTGTTTCAATCACAGAAGTGGCCATTATATGGTA AGGGTTGCTTGACTGTGTTGAGCATCCATGTCAGAGGATGCATTGATGCGAGGCTATGTCACCTTATGCT TGCAGGGTCAACGGCAGCTGTTTTGCATGGCCCGTGCCATGTTGCGGCGTAGCCGTATCCTGATGTTGGA TGAGGCTACTGCCAGTGTTGACCCCGAGACAGACAGCTTGATTCAGGCTGCCATCAGGTCGGCGTTCCAG GAGTGTACACTGCTCACCATTGCACACAGGCTGAACACTATCATGGATTCAGACAGGTGGGTCACTAGCC GCGTAGCACAGCACAGCACAGCACAGCACAATACAGCACGATTAACATGTGGTCAAGCCTGTCAACAGAT GGGCCATTTGTGCATGTCAGTATCCATCTGCATGAATGTGCTCAGATACACTCGCTGTCGCTGCATGTGT GGGCTTGTCATGCTTTCAGATGCAACCAGGCCAGCAGGCTCATCTGGCATGAACCCTGTTTATTTTGTAG TGGATATACAGTATTGACAGCAGGTGGAGGAAATTCTGGTCGTGACTGTCTAGTGTACAGTGGTGTGCCT AAGTCTTGCGTTTTGTCTCTTCTTTCAGGGTACTTGTGCTAGACGGTGGAGTGGTGGCTGAGAATGATCA ACCACACCACCTGCTGCAAAACAATAATGGCCTGTTCACACAAATGGTGGACCAGACTGGTAAACAGAGC AGTACATACCTTAAACAGGTGGCACGCTCTGCCACAACGAGCAGGCAGACTGCCAGGCGGCGGATGGCTG TTGCACACCATGACACCGCAGAAATAACTGATATGGCTGGTCTGCGGAGTGAGAGTACTGTGGGAGTAGT ACCACAACTAAGCCCTACTATGGTAAGTAGGGATATAGTTGTGTGAAGAATGGATGAATGTGCTGGCATG GGTATTGCAAAAAGCTACACAGTGCAAGGTGTCCCCGCATGTAACAGCTCACTTGCCGAAACCAGGCCTT CTTGCCAGATCAACAGACTGCCTGTTGTTATATAAGTTGGTTTGAGGTGCCACCAAAGAGCGAGCACTCA ATCACTCAATCATCAGAAATAGGTTTCCACCTCCCTGCACTGCCCTGTTCCAGCCCCCTTGCTTGCCGTG CATGTGAGATGTGGTGTTATGCAGCTAAAGCAGTTTGCAGGGCTCAGGGTACAGGGTAACGCAGGAGTGC TGCTGCGGCATCATACCTTGTTGTGCCATGTCCTGCAGGACCCACTGGGCCCAGCTGTTGAGGCAGCTCC TGCTCGTTTGACAACAGAGGTGTCACAGCTGCGCCGCAACATCGACGCGGGTGATGTTGCTCTGACATCA CCCACTGTTGCTCTTTGA SEQ ID NO: 11, gene sequence (starting from ATG) Cz08g16130 ATGAAGGCTGACGCAGATGTCTGCATCAAGGACGAGACCGTTGTGGACATGAAACGGCTTGAACTGGACA ACGGCATGCCGGGAGTAGTCAGTGGAGGGTCTCACCACCTCCCTGCAAAGCGCGGCAGCAGTTTTGGGCC GTCTGGGATGACCATCAGTGTGAAGGTAACAAGACGCGACCAGCCATTGAAAGTCCATCATGCGGCAGCT GTGCATGGTGTGTCTCATAGTAGGTAGTACCCCGTGGCCTGGAACGTTTGGAAACTGAAATTGGCTGTCT GACTGCATAACCAACAGGTAGTTCAAGGCCAGCTGCTGGCAATGTGCAGGCAATACCTTGCAGCTGCAGC GTTTAAATGATCTACCAGCCAACACTCTCCAGTGTCTGTTCATCAGCAACATTGAGCAGCATACATAACA CAGCAACAATGCTGCATGGCTCACATGTGCTGTGCTATGCAGGACCTCACCTTCTCTGTCAAGTCAAACA TTGAAAAGGGCAAGACCGTACACTTGTTAAAGAATGTTACAGGCTTCTTTGAGCCTAACAAGATGTCCGC CTTGGTGAGTCACATGACAAACATAAAATCACTTAAAACTGTTATGTGGCAGCTGCCGGATGCATGGCAT AGCAGTGTGCAGTACATGTTGGCCTCCTACACGCAGCAATTATGTGGGGCAATAGCTGGCTGACAGGAAG ACTGCATGCCAACAACCATGTACTTGCCTCTAACAAGAAACTGTCCACTAGTGTGTCAACACAGTTCCAA TTGTGTGATGTTGCTAACAGATGGGTCCCAGTGGCTCTGGCAAGACCACCCTGCTGGATATCCTGGCTGG CAGAAAGACCTCAGGCAAGACAGAGGGAACCATACTGTTTGCAGGCAACAAACCAACACGGCAGTTCCTG CGACGCTATACAGGATATGTGGAGCAGTTTGGTAAGAAGACGGCGGTTGCAAGCATGTGGTTGCTAGCCT GCAACAAGTTGGTGCTTGCAGAATTTGGATGTAGCAACCTTGGCCTTTGGACTTGACAGGAATATTGTGG CGTGCAGCATGGCAGTGACAGCTCTGTCATGTCAAGCAAAAGAGACAAGAGAGCAAGTTCTGTGAACACA CATACACTACACAGCATGACTGACTGTGTCATGTTTGTTCATTTTGCAGATACTCTGCTACCAACTCTTA CAGTAGAAGAGATGCTCATGTACACAGCTGAGCTCAAGCGGCCCATCTCAGAACCCTTATCTGAGAAGAA GGCAGCAGTAGATGAACTTATAGACAAATTGGCGCTGGAGAGTTGCAGGTAATGCTGTTTGATATACTTC AAGCAGTTGAGCAATCGCATGAACATGACTGGCGTCAAGACCCATGGAAACCTGCATTGTTGCTGCTAAG AACAGCTCATTTTACAGTATTGCTGCTAGTACGGCTAGATTGTGTATGAAGTCATGTACACGCATTCCAA GGTGCCTTGCAGGTAGTACTGTCCAATTGCAGGCTGATGGCTGCTTTATCCAGTATGCCTTTCAACCACA ACGCTGCCCTGTGTGTTCCAGGAAGGTGCCAATTGGTAGTTCCATGAGCAAAGGCATCAGCGGTGGTCAG GCCAAAAGGACCAACATCGGTATCGCACTGATTACTAATCCACGTGTGTTGTTCCTTGATGAGCCCACCA GTGGTTTGGACAGCTTCACGTCCAATGAGGTGAGTCATCAGTATGACAGCTGCCAGCAGAACCAGACCAG GGATATAAGTGGCGCCAATGCCCGTAATGGCAGCCCCAAAACTGCTGCAGCAGAATATACTCAGCAGCAG CTGCATTGGGATGCAAAGCCAGTGGCAGTAGGACAGGCTGTAATGTTTGGGCAGTGTTTTATGGTGTAGC CCCATCGCTTCCAAGTCTTGTGAATGCATTGACCAAGGAAGAGGTGTTCAGTGTGCAATTTGACATTGCT TGCCATGCAGGTGATGACCGTGGTGAAAGCCCTGGTTTCCGACGGTGTCACGATTGTCGCCACCATTCAT TCTCCAACCGCCTACGCCTTCAACCTGTTTGACAAGTTGATGATGCTCGTCAAAGGCAGGGTGGTGTATT TTGGTGCTCAAGGCAAACCAGCACTTGAGTATGTGCGCACCCAGTGCCCGCAAATCAAGGAGCAGTCAAG TGGGTATGGCAGTGATGCCGAGTGGTTGGTGGACCTGTTCACTGAGGCAGATAGGATGGGTAAAGGCGGG GAGTTTGCAGACGCTTATGATGTATCGCAGCTCAAAAAGGTGAGCAGGTTGCAAGCAGGTTGCCTTGCTG TGAGTACATGTACTGGTGGGTGGTTGAGATGGACAAATGGGGGAGGTGGGCGTGCACCAGGGGACGGGGG CATGTGGGAAGAGGGGGAACCTGCCGAATGTATCCGACAGTGTTGTTGTTTAGCATGAGATGCATGCCTA TACACTGCACATGTAGGAATAGAGCACTTGCTGATGGGTTGCTGGGCTCATTTTGATGCATACTACCTGC AGCACCCCATCATGCATGCAAATGTATGAACCAGTATCTGCTCTGCTTGTGTATGCAGGACAACGACTAC ATTGTGGACAGCCTCTGTGCGCAGAAGCATGTTCTGCCTGCTCATGTTCAGCAGGAGTTGTCCGTTAAGA CAGAGACAGTCACACCCTGGTGGTGGGGTATCAAAACACTCATCAAGGTGAGAGGGTTAACTGTGCCAAC AACACAAACAGCAGACCACGGCCATGTTGATTGATGCAGGCTTGGTTGCATGGTTGCTGCTGCACTGGGC TGAGCACTGCAAACTCCATGGTTGGTTGTCAGAGACATAGTGGCAGTGACAACTGCACTTCATAGCATGC TTGCTACAGAATGATGCAGGTAGTCACAAGCAGCACTCACAGTATCATGTGTGCTGTTTGGTATTAATCA CGTGTGTACCTCATTGCCGTTGCAGTACCGCACCACTCATAACTACCGTGATGCTGCTTTCCTGGGGCCC CGCATCGGCGATAAGCTGCTGATTGGGCTGTTGATTATGACCCTGTACCTGGGTATTGGAGATGACTTTG CACCAGACAACGTCATCAATATTGCAGCTGTGAACTTCATGTTTGTCACAATGCCAGCTTTTGGAGCAGC TGCTTATGTGCCTGCCATTGTACTCGGTGAGTGGGATCATTGTCCAGCAGTGGCCACAGACAGTCTCAGC AGCTTCACAGTGCATGCCAACTGTGCGGTCAGGTGGTACAGCAATAAGCAACCACCTCAGCATTTACTGC AGGCAATCATCTGCCAGAAGTTCTGCGATGTGCGTGTTGGCGTTGCACATGTCAGCAGTCTGCATGCAAG TGCTAGGACTGAGGCCGTACATGCTGTTGTTGTGCACTTGCTGTTGCGAGTAAACTTCTTCAAATGGTCT TTTCCTGCAGCCTGTCAGTCAGTGCCATGCCCACAATTCTTGCTGTTGCAGAGCGCAACTTGTTCTGTCG TGAGCGTAATGACGGCGTGTATCGTGTTATCACATACCTGATGGCAAAAATGTTGGATGAACTCATGATT GCAGCTGTAGCAAGCTGTGTGATTGCAGCCATAGCATTCTATGGCATTCAACTTCAGGGGGAGTTCGTCC TGTTTTGGCTGGTGTATTACATAGTGCTATGCACGGGCATAGGTAGGGTGCACAGTTTGTGCATATGCAA CGGCATGCAATACTCTTGCTCAACAAAGCGCTGAGCCTGCCTGTTTCTGGTGGTATGGTTCATGTTAACC ACTTGGTGCCTCTTACATAAACTGCTAGCAATGCTGGATTGGAGCAAATGATCCAGCTGGACACCCAATA TCATATGCTGCCAATGAATCATATCCGTTTCACCTGTGTACTAAGCTCTGCTGCTGCTGCTGCAATGACT ACTGCAGTGCTCGCCTACTTTGTGGCCGCTCTCAGCCCAAACATGGATGTTGCTAATGCTGCCCTACCCA CCTATGTGACCAGCCTGCTGTTCTTTGGCGGGTTCCTGTTCACTTTTGATAAGATGCCTGTGTGGTGGAA GTGGTACTCCTACATTGATGTGATCAGGTAAGGCCAACACAGGGTGTGGTTGCATCAATAACGGCAGCTG ATATTGCTGATGATGCTGCCTGGTACCATGCAGCATATGTTGGGACGCCAGGCATAAGCTAATACAATCC CCAACTTGCAGCTTGGCATGATGGCCTGCAGCTACTTGTATCTGTGTGCTTGATTACCGCCTTCACCAGT AACCCTATCTGTGCTTGTGTTACTAGCCCGTTAAAGAGTGTGTAGCTAGTAACACAGTTGTTAAGGTCCT GCTTGTTTCTTGTGCAGATATGCTTGGACAGCAATCATGGTGAACCAGTTTGAAGGTCGGGATGCACAGA TGTTCTCAGGCCAGACAGTACTGCAGTATTATGGCATTGAGGGACAAAACAAGTGGGCCAACCTTGGCTA CACAGCATGCTTCTTCTTCTTTTTCACCTTCTGTGCTTGGGTAACACTGAGTGTGAAGAAGTATCAGAGG CGATAG SEQ ID NO: 12 gene sequence (starting from ATG) Cz09g27180 ATGCAGCTGCAATTCACCAATCAAGTGGGCCAGGCACGGCTAAGGGCTGCCGTTCCCCGACCAGGGCACA AGCATGCCGGTGTACGTGCAAGGCCTTTCCAAGCTGCAAAACCAGGGTGCAGACCACCAACAATCTTAGC CAGCTCTGCGGCTCTGTCATCCATTGACTATACCTATTCAAGCAGCGTGAGCGATGCGAAGTCTCGTCCC TTCCCTGCCGTCGCTCCCTTGCCATCCCCACCACGAACAGCTGACTTGGGAAACGTGTTGCCATACCTTG CCAAATTGGCCGTTGGAGAACGGCAGCTTCTATGGCGCTTCGGGGTGGCATTAATTTGCATGGTGACCTC CAAATTAGCAGGCAAGCGTCTGTAAACATGCTTCATTCGGTGACGGGGGGTGATGGGCTTTGGTACACCC ACTGCTGCTTGGATGCACACCTGCATGCGTCGTCTGTGTACATGCGACTAGCCGCATGTGCCTTCATTAT GCTATACAGCACTGTACCAGTATGCTGCAGCTTAAGCCTTACAGAAGTGGCATTGCCGTAGCCTTGGATT TATGATACACCTGTTCATGGTCGACACACTCAACCTTGTTGAACCAATGCTTTGTCTTTGTTGTTCTGTA GGTTTGGCAGGCCCAGTACTGTTGCGTGAAGCTGTAAATGCAGTTGGCGAGCAAGCAACCGCATCCTTAC GTCCTGCTGTGCATGCTGTTGTCTGCTACGGTTTATGTGGTGTCTTAGGGACTCTGGCAAAGGAGCTGCA GCACCCTACATTTGCACCTGTGTCTCAGGCTGTAGCAAGGCGTGTTGCCTACCACACTTTTGCACATGTG CTGGACTTGGATATCAAGTTTCATCTGGAGCGTAGGACTGGCCGGCTGTCGAGGATACTTGAGCGTGGTG AGCAACAGACAAACACCAGCAGCAGCAGCTTTGGCAGCATTCTGTAGCATGCTAGTGGTTCATTCAATTT GTGTACATGTACTCACAAAGTCAAGTGGCACTTTGTTTTGCTAATGGTATGCTCGTCCCTGCATGGTGCT GACACCCTGGACTGTAGACTTAAGTACGCCCTAGCCGTGCAGCCTCAGGTTCACTGCAATTTTAACTGCA ATTCACAAGCCGCACCTAGCATGTCTTCACTTTGTGTGCCAGGTACACGTAGTGTGCAGATGCTATACCG AGCAGTGCTGTTCACCTTCATACCAACAGCATTGGAGTTTGCCTTTGTCATCGGCTTGCTGGGGACTCAG TTCAGTTCAACTGTAGCTGGACTTGTGGCAGTTACATTTGTAGCCTATGTGGCATGGACACTAGCAATGA CACAGGTGGGGCTATGGGCGCCTCACTTAGGCTCCATGGTATAATGTCAGTGTGTATGATGCAACAGTGC ACTGAAGTAAAGAGCACTGCACATGCAGAACAGTGTAGTAGTGCACACCCTGTAAGTTGGGGTACTACCT ACAACCCGAAATACAAATTCACTCTAATGTAATCTGTGTACAATGTAACAATCAACCCCTCCCATGTTCC ACCTGGCAGAGTGCTGTTGAGGTACGCAAGCAGGTCAACACCCTGGACAACCTCACCACCAGCAAGGCAG TAGATGCACTGTTGAATGCTGAAACAGTCACACTGTTCAACAACCAAGCGCTGGAAGTTCAGCAGTATGA CCACTACCTCAGAGGCTTTCAAAGAGCAGCAATACAGACTGAACGCTTGAGTGCGCTTCTCAACGCTGGC CAATCTGCCATCCTAACCATTGGACTGATGCTGGTGCTGATTGCTGCTCTTGTGAGCGCACCTGCCACTC GCCCTGTGACTGCCGGTGATTTGGTGCTGTTGCAAGGCTTGCTGCTACAGCTCTGGTCTCCTCTGCAATT CCTTGGTTGGTTTTACAGGTAAGGTTGACTCAAGAAGCACTCCACCAGCTGATGTGCATGACTAGTGACA CAATACAGAACATGCATTGACTGGTGCCGCAGTGCATGATATCCCCTATGAGCACTGTTGCAACTGTGCA TGTAATTTATTTATTTATCCATATTCGTCCACAGTTGACATCACTTGCCTGAACAATAAGTGCACAATGA TGCATATGTCATGAGTGATCCTTGCAACAAGCTTGCCTTCTGGTGGTTGGTACTGAGCATGTTGTTGGGT GTCAATGCCCCCTGGCCACCAATCATGACATTTGACAGCAGCACAGGTGGTGTGCATTTGAGAACTGCGT TTTTCACCCTTGAGGAACATCAGTGCGCTTTGCATGCTACAATTGGCTGCCTCACAGCGATAGTGTTAGC TGCTGCCTACTTACCCCTACCTGACACTGTATGCTCACATTGCTGTGCTGTGCATGCTTTGCCACTGCCA GGGAGTTGAGACAGTCCTTGGTTGACATGGAGGAATTCTTTGAGATACTTCAAACCCAGAGCCAGCTGCC CGATGGCCACCTGTCATTACCCAACACGCCCCCATGCATGGTGCGAAACATTGCAGCACAAACAAGCAAT AGCACAAGCCGCAATGGCAACAGCAGCCAGCGTTTGGGTAGTAACACTGTCAGCAGCACAGGCACGTCCC CACATCACAGCCACTCCCAGCAGCAGCAGCAACCGGATGATCCTACTGCCAGTTATCATGAGATACCTTA TGATGCGGCATGCATCTCAGGCTTTGGGTTGGAGGTTGAACTGAAAGATGTTCACTTTGGTTATCATCCT GATAGACAAGTGCTGCGTGGTGTCACACTGCGCATACCACCCGGTCAATCAGTGGCCATCGTGGGCTCGT CAGGGTCAGGCAAATCAACCATACTGAAGTTAGTCACCCGGTTGTATGACGTAACCACCGGCAGCGTGGA GGTGAATGGTGTGGATATTAAAGATCTGACGAGAGATAGTTTGAGGGCAGCTGTGGCAGTTGTCCCTCAG GACACTGTGCTGTTCAATGACACTATACTGCAAAACATCAGGTAAGCAGTAGTTGCCCTTGAGCTGCAGT CATCACCTGCAGCTGGCACAGTCAGTGGTATTGTGCATGCACCGTCATGTGGTATGACATGCATTCTACT CAACTTCATCTGTAGCTTGTTTCCCAGAAAGTTTCGAATACGTGGTTCAATCTGTGTGAACCTGGTATGT TGCTGTGCTGCCAGAGCATGCATATGCATGCATGCATATGAGGGTGGGGTGGTCTTCTGCCCAGTGCAAC ATGCAGGGTCTAGCGTATACTATGTTACAGTAATAGCTGGTGTGCTGTAGATGCTGGTCTTGCTAGTTTC AGCCGCTTGGTCTTAATTGGACCAGGTCTTGGTTCCAGGGGCAACCAACATTCCTGCCCTGTTGTGCTGT TTCTCATCAGGTACGGTCGTCCAGAGTCAACAGATGATGAGGTCATTCGCGCAGCTCACTTGGCCCACCT CCATGATGCTGTTGTCAAGATGCCTGAGGGCTACAAGACAGTAGTGGGTGAGAGAGGTCTAAAACTCTCT GGGGGTGAGAAGCAGCGCGTTGCGATTGCGCGTGCGTTCCTGCGAGCACCACGTCTGCTGATTTGTGATG AGGCTACGAGTGCGCTTGACAGTGCTACTGAGGCATCCATCATGAACTCACTGAATGAACTGGCTCAGGT AGGACAGGGTTGATGGAACAGGGGAAATGGATTGAAATGGAAATGGGTATTGGAGGGAGGTGGAACAGGG GGAATGGAGTGAGGTGGATCATGGTTCAGTGATGAAGAGGGATGTATTGTAGTATGTTCAGTGCTCTCTC ACACATATTCATGTTCAGCTGATTGCATTCATCGCCTGTTTGAGTACCAATGATTTGCACGTTGTGCAAT TGCGCTGTGTTGCCAATGCTGCACACTGCTGGTGTTCGGATAGTGAACATGCAACAGCAACTGCATGCAC AGTGCATGCTACAGTATGTTATCCCTGCAATTGTTGTTGACCATCAAAGAGTGCTGCTTAACAGCATGCA GTGTGAGCGCCAGATCAAAGTGGCCTTGCATGTACATACTGAGTGCCCATGTGTATCTGCAGGGTCGCAC AAGCCTCTTTGTTGCTCACCGGCTATCCACCATCCGCAACTGTGATCGCATTGTGGTGTTATCGGCTGGT GTGGTGGTAGAGGAGGGCACACACGATCAGCTGATGTCACGTGGTGCAGTGTACAGAGACATGTGGGAGA TGCAAGCAAAGGAAGCCAGTAGGGGAAATGGCGTTGCAGAGGGGACCACCTCAGACAGTGAAGATGGGGA GCGAACAATGGAACCCCTGCCAGCTGCATTGACAGCTAAGTCCTTGAATTGA SEQ ID NO: 13, gene sequence (starting from ATG) Cz10g28330 ATGATGCACACCTCGTCCTCAACATTGTACCCACTCGGTATTATAAGTTTCCTGGTCATTGTTGTGCCAT TAGTCATGCACGTGCTGTCTTCCCGCCGGCTGGACCTGGACCTGCTGCCCAGCCCTCCTGCTGTGCTGCT GTTGGGACACATCCAGCTGGCACTCCATGTTAAAGACATGCACCTGCAGTTTCTACGCTGGCATAACCGC TTTGGCAAGCTGTTGCGCATCAGAGTGCTGCAGCAGGATATGGTGCTCATTGCTGACCCTGCCCTGGCGT CTGAGGTGCTCACGTTGGGTCCAAACTACTGTGCACGGCGGCCAGCAGACTATGCCACCTTCAATGTGGT GAGTTTCAGTGGGTGCACAACTGTGCATGTCAGTCCATGGTGCAAGCAAGCCATTGGGGGGCTGTAGCAT GCAATGCATGATGCAACGCAGTGTGCAAATCAGACTTTTGAGTATGGTTATGATGCAACTCTGCATTAGC AGTAGCACATCAAATCACATGACTTCTAACTGACCAGACCAGTCAGTACACAACAAGCCAGCCAGCCTAC ACAACAGAGTGTCATGGCATGTGCTGTGTTCATCTGGCACCTGACAGTACCTGGCCTTTGCAGCAACATG CAATGCCTGCAGCATTTTACAGCTGGATTGTAAGACCACCCTTGGCAGTTTGACGGGTGCATGCAGTGCT TGCCAGCTGGGTTGTGTCACACTTGCGCCTTGGTTCCTCATGTCCTTATGATGCATCAGTTGTCCCTCAG GCTCTCACCACCCTGTTGCTGTTTGCTTGTTGTTGTCATGACAGATTCATGGCCTGTCAGCACGGCCCTC CATCCTTACTCATCAAGATGAGGCATGGTGGAAGGCTGTCAGGAGAGCAATAGCACCCGCCTACACACCT GCTGCCACCAGGTAACAATAGCAGTCAGCAGCATGCTGCAGCTGCCAGCTGGCTGCTGTAGGTGTGCAGT TACGCAGCAGTCCATTGTGTATGCAGCAGCTGATTGTGTATGCAGCAGCTGTCTCCATTTTGATTACTTC ATTGCTGTCTTGTGTGCTGTGCTGCAGGGAGTTGCACAGCCTCATGGTATCCACCATGGCTCGTGTCTGT GAGCGTATCAATGCCAACCTGCACAGTGCTGCCAACAACGGCAATAGCACTAGCACCAGCAGCAGCGATA GCACTGGTAGTCCAGCAGGTATACGCATGGATGAGGAGGTGCTGGTAGCTGTGCTGGAGATCCTGTTGCA TGGTTCACTGCGGTTACCACCACAAGCCATGTCATCATCAGATGTGCGGCGCACTGCACAGGTGGCCCCC AAGTTAGTGACCATTGCCAACAGCTTTGTGTCACTGCCAGGCAAGCAGTGGGTGTACACCACATTGCCTT TCATTTGCAAGGTGGGTGAGCCAACTGCTAAGAGGCAGTGCTCTTGGGTTAGGTGATAGCGGTTGTACTG GTGGCCACCAGGGTGGTTGCTTTTGGTGCTTGGGGTGGTGGTTGGGATGGGGCATGCAGGTGGACAGATG TGTCTGGTGGCTGTGATTTGACAGGCACACATGACAATGTGACATAATGTTGACCTGCATGGGGAACACA GTTCCTTCAGGTTCTGTGGTCATGTGTGATGTTGAAGGAATGCATGCTTGCACACAGAAAACAGATCCAT GTGGGTAATATCTGCTATCACAGGGGTATTGCGGGCAGACCCAGCACTAGGTATGTTCGTAGCATCATGC AAGTGTGCTCAGCTGCCACACCATGACATGTTGTGTTGCTACGCTGTTGTCTGCAGGAAGCTCGTGTCAT GCATCAGGGCCGAACCCACATGACTAATATCACACGTCAAATCTATAATCACATCAGCGCCAAATACACT GTGAATGGTGTACCGTCTGCAAAGAGTGATGTGGGAGGCTCTGAAAAGAGTGACACATCTCTCGCCTCCT GCTTGATGCGCCTGGCCCACCCTCAACACACGCCTGGTCAAACATTCACCCAGGATGATATCATGGCTGA GATTGCCATCAACATCATAGGACAGGGATCCGTACCATGGACAGTATCTTGGGCACTGTAAGTGCGTCTG TACATGCCACATTGTGCTGCTACTGCCAGTTAACTACTGCATTGAGGCTCGTAGGTTGTGTTGCTGGCAT GGCAGGTTGTTGTTCAGAAGTTTCTGCTACTTGCTGCTACTTGCTGCAACCCAAATATGCACACAGGTTT TATGTGCTTTTTGACATGGAGAAGGATTGACACCATTGATTGCAGCAAAGCAACTTACAAATTCAGCACT TACACAGTATTTACATCGGTTGCCTAGATCAAGTCATACATTTCGCAATGGACACTCAAGCTTTGTGGTT GTGTTATTCATACAGGTTCCAGCTGACTCAGCGGCCTGATGTGGAGAGCCGATTGCTGTCAGAGCTACAG GGCCTGGGGTTACCCTGTGATGGTGACCTGGCGGCAGCATGTGATGCCATTAGTCATCCGGAGTGTCTCA GGGACACCCCCTACCTGACAGCTGTCATTAATGAGGTGATGCGCATGTACCCAGCTGGTGTGTCAGCAGC TCCTAGGTAGGGTACAGGGTTTCAGGGCTTAGTATTTCAGGGTTTACGCTTTGTACTTGCATTGTGTTTT ACGCCATGGATTGCTGCAGCTTGCTGTTATCCATCACTTACGTGCAGGGTGCTGCCAGGCACAGAACTGT AATGCAGGGAGGTCTGAAGACGCGCTTAGACTGTGGCGTGTGATTGATTGTGTTGTTGGCAAAGGTGTGA GGTGTCAGGCTGAACCCATTGCATGCTTTCTGAGCATGAATGGAAGATGTCAAATCTGACTACTGTTGTG CATTTGTCTTTCTTGCTGCTCCTACAGGTTGACAGAACAGCCCATCAAGCTTGGAGGGTATCGCATACCT GCTGGCGTTATGGTCTTCCCAAATTTATTCACCATCATGAATTATAGAGGTAACTGGCAGCAGCCAGATC AGTTTTACCCAGATCGCTGGCTGCAACCAAACTCATCAGTAGACCCCTCTTCAGGCGCCCCAAGGTTTAT CCCCTTCAGCATCGGGCCAAAGATTTGCATAGCACAACATCTGGCCATAATGCAGTGTAAGATTATGTTG ACATTGTTAGTCAGCTGCTTGAAATTCCAGCTGGCACCCACAATGGGGGGAATTGAGGGGGTGATGAAAC GGGTAGAGGCGACGTTGGAGTTGCGGGTGCAGGGGGGCTTGTGGTTTACTGCAGAGATCAGGAGGTCTAA CAATGTAAAATGA SEQ ID NO: 14, gene sequence (starting from ATG) Cz11g14160 ATGGATGGACTTGCGATCGCCATGGGCTGTGCCGCGGCGGTGCTGGTGTGCTATACGGTGTTGCCATTCC TGTGCAGCCTTGCACTAGCCATCTGGAGGTTTCATACAAGCAAGATACCCGGGCCTCCAGCAAAAGACTT CATCCTGGGTGAGCTTCATTGCAACATTTTTGCATGCATGCGCACGTATTGCATGCATGTGCAAATTCAT GTGCAAGTTGTTGAACACTGCTTTTGCTGTGGTGGTAAATGTTACAATCACGTTGAGCTATATAACACTT ACCTTGTTTGCCTCAGGCCATGCTGCGGCTCACTTGAATGACAAGGCTCCTTTTATTTTCAAAAGATGGG CTGAGAAATATGGCAAACTTTACAAGGTTCGGATGGCCGACAACTTCGCCGTGGTGCTGACCGATCCCGA AGTCATACAGTCCATAGCTCGCAAAATACAAAAGGTAAGTAGCTCAGCCGCTGCCAGCACCATTCTGCGC ATCATCCACGTTAATCACACCCAACCGTCACCCAACCAGGTGATGAATACTGAATACTGCGCATGTACTG CAGAATGAATCAGTTCATTGATTAGCTAGGAACCAAGTTGTAAACCTCTGCTTCTTGCTGTCAGGCGCTC AATTGGCGCACTTTGTAAACGCACTGTCTTGGTCCAGCCAAGCTAGTGTCAAACAGCTACACCCCTTGAT GCAATTCTTGATGCGTTTGTGAGGAATTTGCATTTATGACTCCTTACTCTGCGCCCTGCATGCAGCCTAC CAAATCGTACAGTAGTCTTGAGCTGGGCACGTTCCCCAAGGATCACAACATCTTGACCGCTCCAGATGGC CCACACTGGAAAGCAGTAAGACAAGGCATAACACCAGCATTCAGTGTAGCAAACTTGAAGCAGGTACTGA CGCTAAGTCATGTAGAAGCAGTGCAGCACATCAGCACTGACGCACATCAGAGTTGACATTCAGTGCATTA CCATAGTACAAGTGTCTCCATGTCAGTGTCACTGCGAGCAGCATGGCTGCACAAGGGCATCTCTGCTCAA GCTGTTGTTGTTGTATACAAATGGTACTGTTAACTTGGTAGAGCGCAATAACATCCTGCACACTGCACAA CAATTACAATTTTTTCTACCAGTGTTGACGACAGGTCCTCCTATATTTTGCATCGTGTGATGTGCAGCCC TGCTTGAGACGTTTGATGTAGTGATGAGGCTGCTCTTGTTGCTCTGCTCAGACACTGCTGCTTGTAATGG TGGAGCATTGTACTCACTGTGAGCATAACACCTTATGGTCGTGCAACTGCTGGTGATTGCAATGCCATGC ATTAGTTGCGGTCATAAACTGTGAGCCTCATGATGTCCCTGCTTGCCGCAGGTATTCCCCTGGTTGTGCC ATGTCACCAAGGTGGCTGCAGCTAAACTCCAGGACACAGGCCCTTCCACCAGCTTTGACATTTCTGACAT TGCCAAGCGGATCACCAGTGATGTCATTGGGCAGCTGCTGTATGCGGAAGACCTGGGAGCCATAGCATAT AGGTGGGTTGTGTGGTAGGGTTACCACTACTGATCAATGTCTAGTGGCTGGTAACTGGTGTGCAAAAGCA GTCACATCACGCTGTTGATGCAAACAAATGCTGCTGTGTATGAAGTGTTACAGCACTTACCTGTATCAAG AGCCAGCCTATGGGGTCAGATATAGCGCTTGCACAACAACACACTCTGGTGTGCTGCTATGCACATGTAC ATACTGAAGTATCATTGTTGTGCAGGCCCAGTGAATACCTGGAGCTGTTCCAGATCGGCATCACTGCAGC TCATAAGAGCCTTGGGAGACCTTTACGGCTGTATGCCATCTGGGATCCTGAGGTGAGGCGTCAGAACCGC GCAATAAACCGTCTTGACCAGTAG cDNA Sequences: SEQ ID NO: 15 cDNA sequence starting from ATG; Cz02g29020 ATGGAAGCAGCACTGACCACTGCCGTAGATGCAGCGGCAGCGTTACGAAACAACATAGGCTTCTTGCAAA GAAGCCAGATAGCAATCGTAGCAGCGTTCCTGTCAGCAATATACCTCAAACTGGTTGTGTCAAGGATAGC ACCTGGCTGGAAAAGCTTAGCACTTGTGGCTCCGCTGCTTGCATTGAACCTGTGGCTCCCGCTACTCTTT CATATGAGAGAGGAGGTCGTCAGCAGGACTTGCGTGGTATTCCTAATATCTTGGCTCGGCAGCTTCAAGG CAATTGGTATGTGCCTCAATAGGGGGCCACTGGCTGCTGATTGGAGTGTAGCACAGATGATCTTCCTGTA TGTTGCACCAGTACTACCCCGACAAGATGCTGGCGGCCCTGTCAAAGCAGGCCGGCTGCAGGACAGTGCT GGCACCTGGGGAACTCTTGCGGCATCATTTGTGATCAACACCACAATTCTTGGTGTTGTTTCTTACCTAC TGGTGGTGATGGACATGCCAAAAGTAGTCAAGATATACATCTACGCATTGGGTTTATATGGCTTTGTCTC GTTTATTATGGAGGGTCCAGCAGCCATCATTGTGGGCTTGCTGAGGATCGAACTTGTGCCTCCCTTTGAT AAACCATGGCTGGCGACTAGTTTAGCTGACTTCTGGGGTCGGCGTTGGAACAACTCCACTAGTTTGTTAC TACGGTTCCTGGTGTATGATCCCATCATTGAAGGCAGGTTGGTGAAGAAGGCCAAACAAGAGGATCCACA GCAACCTGCTCCAAGCAAGCAAGTCAGCAACAGCATGAGGCTGACTGCCATGCTTGCTACGTTTGCATTG AGTGGGCTGATCCATGAAGCCATCTTGGCGTATGTGAATCGGCCATACTACCCAGGACCCTGGTTCATGT TCTTCTTCATTCAGGGGCCCTTGTTGGCAGTGGAGATGCGGGTACACAAGCATCTGAGGGCAACACACAA GCAGCTGCCATTCACTGCTGCATGGACCTTGACAACAGCATGTCTGCTGTTAACAGCTTACATGTTTTTC TTCCCTCCCATCGAAGACTGGACAGACCTTGCACCAAGAATAGCTCAAGCAGTCACCAGCAACTTCCAAG CAGTGTTAGGCCCGCTTCAGGGGCTGGGGCGCCAGTACTGGGCTGTAGCAGCTGGGTTGGTGCAACAGTC AGCCATTTGA SEQ ID NO: 16 cDNA sequence starting from ATG; Cz04g21110 ATGCCGGTACCTGTGTCCACAACCCACCCAAATGGCAAGGGAATGCGCGCAGCCAACCTCACGGAGTTAC CCACAGAGGGGCCTTTTTCACGATCGCTGAAGCAGAAAGAGGATAGTGCCAACACAGGAGCAGATGCAAT AGCATTGCAAACAAACGGCACTGCTGACGCCTCGCTTGATGCCAATGCACCATCACCATTGCCAGAGCCG GGACCATCAGTGCTGGTTCATCATCTAGACTTCACATACCCAGGGTTAGATGGCCGCCCTGTGCCTGGTC AACCTCCTCTGATAACCGACATGTGCCTGACGCTACAGCCGGGCAGCAGGTGCTTGCTGATTGGCGCTAA CGGTGCAGGCAAAACAACGCTGCTCAAAATACTGGGTGGCAAGCATATGGTACCCAAGGATGCTGTTCAT ATACTGGGTGCACCACCCTTCCATGACACACATCTCACCACATCAGGGCACCTGTCATACATTGGTGGTA ATTGGACTCGTGATATTGCATTCGCAGGATTAAGCATACCGCTAACGGGTGACTTCCCTGCTTCACGGAT GATTGATGCCATCCCAGAGGTTGACCCCGCACGTAAAGAACGCCTGATCAAGGTGCTGGACATTGATCCA AACTGGCGCATGCATACGGTGTCAGATGGTCAAAGAAGGCGTGTGCAGATCTGTGTTGGGTTGTTGCGCC CATTCAAGGTGCTGCTTCTGGATGAGATTACCGTGGACCTGGATGTGCTTGGTAGGGCTGATCTGATGCA TTTCCTGGCTGATGAATGCGCCACCAGAGGAGCTAGCATCATATATGCCACACACATATTTGATGGTCTG GAGTTTTGGCCCACACATGTAGCATATGTTGCAAGAGGGCGGCTGCAGATGGTCAAGTCAGCTGAGGAGA TATCTGAGCTCAAGCAGGGCAGGTTGTTAGAGCTTGTCACCAGCCTGTTGAGGGAGGAACGCGAGGCAGT GAAAGCTGCTGGCGTCAGTAAACCACTGGAGTATGATCCCAGTCTAGAAGGACAGGTTACAAACTTCTCA TATGCCTTCAATAATGGTTGGGTTCCTGGAACGTTGAGCACCTCCTTGGCCAAGAGTACTAATGCTGTGA TGCGTAATTGA SEQ ID NO: 17 cDNA sequence starting from ATG; Cz05g17060 ATGCTTCAGAGGATAGTAGGCGGGCTTCAATGCTACGCAAGGCAGCAGGCTGTACCGTCCGTGCAGTGTC CTGAGAACGCCTATTTGTACTATTGGATCATAGGTCTGTCGTTAGCACCTTTACTACAGAGCTTAGCAGA AAACTCCATGAATTTTGAGCTCAGCGTCATCGGCACACGCATGCGCAATGGACTGATGGCAGCCATATAC CGCAAGTGTCTGCGGTTGTCCAACTCAGCCATACAGAGTGAGAGCACCGGCAAGGTGGTCACACTTATGA GTAATGATGCTCAGAAGGTTCAAGATGCCATGATGGCCATCCACACCATCTGGGGTGCACCTGCCCTCAT TGTGGTCATTACCATACTGTTGTACCAACAAGTGGGATGGGCAACCTTTGTGGGACTAGGAATCATGCTT ATCTATGCACCCATGACAGGCAAAGTGTCACGGAAGCTGGTGGTACTGCGTCGCAGCATCATGCAGTGGA CAGATCGCCGCGTGGGCCTCATGAATGAGGTCATCAATGGCATGCAAATGATCAAGTTCTATGCCTGGGA GAACTCCTTCAAGAGGCAGATCCTGCATGCCAGAGACAATGAAGCCAGAATCCTGAAGACTGTGGTGTTG TGGCAGTCATTTTTTGCAATGCTCCTGTTCAGCGGCCCTGTGGCTGTAGCAGTATTCTGTTTTGGTTCCT GGGCATTAGCTGGCTATGGCCTGACAGCAGCGTCAGCCTACACAGCACTGTCACTGTTCAGTCTGTTGCG AATGCCTCTTGTCATGTTGCCTATGATGATTACCATGATCATTAATGCACTGGTGGCCCTGAACCGCATC AGCGCGTTCTTGCTGCGTGGCGAGATACAGACCACCAGCCAGCCAGGTCATGATGGCAACAGCAGTAAAG AGAAACAGGAGACTGTTCAGGAGACTGTTCAGGAGACTGTTCAGGAGACTGTTGAACCTGGAGTGGTGCG GGTGACTGAGGGGACCTTCAGTTGGGACTCTGCTGGCGATCAGGCAGCCCTGCGTGATGTCAACTTCACT GCTGCCCCTAGTTCATTGACCATGGTGGTGGGCAGTGTGGGGTGTGGCAAGAGCAGTCTGCTGTCAGCAT TGATTGGCCAGATGGAGAAGCAAGGAGGAGAGGTGCAGCTTGGAGGCAGGGTTGCCTATGTTGCACAAAC GGCATGGATCATCAACGACATGGTGCAGGAGAACATCCTTTTGGGTGAAGCATTCGATGCTGATCGGTAC CGTATGGCAGTGGAGATCTCTCAGCTAGTGCCTGACCTAGAGCTACTGCCAAATGGTGATCGCACAGAGA TTGGCGACCGCGGCGTCACCCTGTCCGGGGGCCAGAAGCAGCGTGTGTCGATTGCAAGGGCTGTATACAG TGATGCTGATGTGTACCTGTTTGATGATCCTCTCAGTGCTGTTGATTCCCATGTGGGTCGAGCACTGTTT GAGAAGTGCATACGGGGTGTCCTGCGCAACAAGACAGTCATCCTGGTGACTAATGCCCTCCAATATCTGC CCTCTGCGGATAATATTTTGTGGATGGAAGGTGGTGCTGTGAAGGCTCAGGGCAGCTACAGCCAATTGGT GGCCGCTGGCATGAATGTGGCTGAGCTGGTACATGTTGAGCATGAAGCTGAACAGACACATCAGCGGGAT GAGGACCCCAGCAGGCAGGCAGGCACGGGTGTAGTAGCAGAGGAGGCTGACCTTCCTGATGATCATGACA GTACAGGCACGTCTGGCACTGACTCAGCTTACAGTGATGACATTGCCAAAGCTACCATCGTTTCTCATGA TGATGCTGATGGTGATAAAGCAGCCAGGCATGTGAAGGGGTCCTACATCCCAGAGGGAAAGCCATCAAAG TCCATCACGCTAAGGAAGATAGATACTGTAGACAACAGGAATCTGACAGGTATTGAGGCTCGTGAGACGG GCGCTGTGAGTGGGAAGGTGCTCAAGTCATACATCATCGCTGGCGGCGGCTTCATCATTATAGCCTTCGT TGCGCTATTGTTTGCTGCTGAGCAGGGTGCCCGTGTGTTCACGGACACTTGGGTTGGGCTCTGGTTTGGT GATGCGTTCAGGCAGAATGTGTGGTTCTATGTGGGTATCTATGCAGCACTGGGAATACTGTACAGTTTCC TCACTTTCCTCAGGGCACTTCGTTTCAACTACTCAGCCGTCAATGCAGCCCTGTCTCTCCACAACCAGCT GTTGAATCACATCCTGCGCCTGCCCAAATCATTCTTTGACACCAACCCAGCTGGCAGAATTCTCAACAGG TTCAGCAGAGACACTGAAATCATGGACAGCACTGTTGCTACATCAGCGCTCATGTTCTGCAACTGCATGG CGACATTCATCGCCATCCTGATTGTGATCAGTGTTGCAACGAAATGGTTTGCTGTAGCCATCATCCCTAT CACCATCACATACATGCTACTGCAGCGGTATTACATCCCATCCGCTCGTGAGCTGCAGCGCATTGAAAGT GTGTCCCGCAGTCCCATCTACAGCAAATTCAGTGAGGCGCTGGCAGGCGTGCCCACCATCAGGGCTTACA GGAAGGAGCGGTACTTTACAGTCACCAGTGATAAGCTGATGCAGGAGAACGCATACGCTTACATCAGTCA GAGGTCTGCAGCTTCATGGCTGGCCATGAGACTTGATGTTGTGGGTGTGCTCATCCTGACATTGACAGGT GTGTTGTGCATTCAAGGCACCATCAGCCCCGGCCTAGCAGGTCTGTGCCTGGTGTATGCACTGGACCTGA CACGCTACCTGAAGATGGGCACGGCAATGGCATCCAAGACAGAGTCTGATTTCAACAGTGTGGAGCGAAT CATTCAGTACCTGGAGCCTGCACCTGAAGCTGATGCTGACACACCCGCAGATGTGCTGGCCACACTGCCA GCCAACTGGCCTGCTGCTGGCAGCATCAGCGTGAGGGATGTGTGCATGCGCTACCGCCCTGGGCTACCTC TTGTGTTGAAGGGCGTCAGTTTTGACATTGCGCCTGGTGAGAAGGTGGGGCTGGTGGGCAGGACAGGCAG TGGCAAGAGCTCACTGTTCCTAGCACTGTTCAGGATGGCCGAACCAGAGTCTGGCAGTATTCTGATCGAT GGTGTTGACATCCGTACCCTTGGCCTGCACACACTGCGTGCTGCTATGTCTGTCATCCCTCAAGACCCCT TCATGTTCAGCGGTACTGTGCGGCACAACCTGGATCCATTTGACGAACATCCTGACAGTGAGTTGTGGCG CGTGCTAGAAGCGGTTGGGCTGAAGACAGTCATATCTGTACTGGAGGCAAAACTAGAAGCTCCTGTGGTG GACAATGGTGCCAACTTTTCACAGGGTCAACGGCAGCTGTTTTGCATGGCCCGTGCCATGTTGCGGCGTA GCCGTATCCTGATGTTGGATGAGGCTACTGCCAGTGTTGACCCCGAGACAGACAGCTTGATTCAGGCTGC CATCAGGTCGGCGTTCCAGGAGTGTACACTGCTCACCATTGCACACAGGCTGAACACTATCATGGATTCA GACAGGGTACTTGTGCTAGACGGTGGAGTGGTGGCTGAGAATGATCAACCACACCACCTGCTGCAAAACA ATAATGGCCTGTTCACACAAATGGTGGACCAGACTGGTAAACAGAGCAGTACATACCTTAAACAGGTGGC ACGCTCTGCCACAACGAGCAGGCAGACTGCCAGGCGGCGGATGGCTGTTGCACACCATGACACCGCAGAA ATAACTGATATGGCTGGTCTGCGGAGTGAGAGTACTGTGGGAGTAGTACCACAACTAAGCCCTACTATGG ACCCACTGGGCCCAGCTGTTGAGGCAGCTCCTGCTCGTTTGACAACAGAGGTGTCACAGCTGCGCCGCAA CATCGACGCGGGTGATGTTGCTCTGACATCACCCACTGTTGCTCTTTGA SEQ ID NO: 18 cDNA sequence starting from ATG; Cz08g16130 ATGAAGGCTGACGCAGATGTCTGCATCAAGGACGAGACCGTTGTGGACATGAAACGGCTTGAACTGGACA ACGGCATGCCGGGAGTAGTCAGTGGAGGGTCTCACCACCTCCCTGCAAAGCGCGGCAGCAGTTTTGGGCC GTCTGGGATGACCATCAGTGTGAAGGACCTCACCTTCTCTGTCAAGTCAAACATTGAAAAGGGCAAGACC GTACACTTGTTAAAGAATGTTACAGGCTTCTTTGAGCCTAACAAGATGTCCGCCTTGATGGGTCCCAGTG GCTCTGGCAAGACCACCCTGCTGGATATCCTGGCTGGCAGAAAGACCTCAGGCAAGACAGAGGGAACCAT ACTGTTTGCAGGCAACAAACCAACACGGCAGTTCCTGCGACGCTATACAGGATATGTGGAGCAGTTTGAT ACTCTGCTACCAACTCTTACAGTAGAAGAGATGCTCATGTACACAGCTGAGCTCAAGCGGCCCATCTCAG AACCCTTATCTGAGAAGAAGGCAGCAGTAGATGAACTTATAGACAAATTGGCGCTGGAGAGTTGCAGGAA GGTGCCAATTGGTAGTTCCATGAGCAAAGGCATCAGCGGTGGTCAGGCCAAAAGGACCAACATCGGTATC GCACTGATTACTAATCCACGTGTGTTGTTCCTTGATGAGCCCACCAGTGGTTTGGACAGCTTCACGTCCA ATGAGGTGATGACCGTGGTGAAAGCCCTGGTTTCCGACGGTGTCACGATTGTCGCCACCATTCATTCTCC AACCGCCTACGCCTTCAACCTGTTTGACAAGTTGATGATGCTCGTCAAAGGCAGGGTGGTGTATTTTGGT GCTCAAGGCAAACCAGCACTTGAGTATGTGCGCACCCAGTGCCCGCAAATCAAGGAGCAGTCAAGTGGGT ATGGCAGTGATGCCGAGTGGTTGGTGGACCTGTTCACTGAGGCAGATAGGATGGGTAAAGGCGGGGAGTT TGCAGACGCTTATGATGTATCGCAGCTCAAAAAGGACAACGACTACATTGTGGACAGCCTCTGTGCGCAG AAGCATGTTCTGCCTGCTCATGTTCAGCAGGAGTTGTCCGTTAAGACAGAGACAGTCACACCCTGGTGGT GGGGTATCAAAACACTCATCAAGTACCGCACCACTCATAACTACCGTGATGCTGCTTTCCTGGGGCCCCG CATCGGCGATAAGCTGCTGATTGGGCTGTTGATTATGACCCTGTACCTGGGTATTGGAGATGACTTTGCA CCAGACAACGTCATCAATATTGCAGCTGTGAACTTCATGTTTGTCACAATGCCAGCTTTTGGAGCAGCTG CTTATGTGCCTGCCATTGTACTCGAGCGCAACTTGTTCTGTCGTGAGCGTAATGACGGCGTGTATCGTGT TATCACATACCTGATGGCAAAAATGTTGGATGAACTCATGATTGCAGCTGTAGCAAGCTGTGTGATTGCA GCCATAGCATTCTATGGCATTCAACTTCAGGGGGAGTTCGTCCTGTTTTGGCTGGTGTATTACATAGTGC TATGCACGGGCATAGTGCTCGCCTACTTTGTGGCCGCTCTCAGCCCAAACATGGATGTTGCTAATGCTGC CCTACCCACCTATGTGACCAGCCTGCTGTTCTTTGGCGGGTTCCTGTTCACTTTTGATAAGATGCCTGTG TGGTGGAAGTGGTACTCCTACATTGATGTGATCAGATATGCTTGGACAGCAATCATGGTGAACCAGTTTG AAGGTCGGGATGCACAGATGTTCTCAGGCCAGACAGTACTGCAGTATTATGGCATTGAGGGACAAAACAA GTGGGCCAACCTTGGCTACACAGCATGCTTCTTCTTCTTTTTCACCTTCTGTGCTTGGGTAACACTGAGT GTGAAGAAGTATCAGAGGCGATAG SEQ ID NO: 19 cDNA sequence starting from ATG; Cz09g27180 ATGCAGCTGCAATTCACCAATCAAGTGGGCCAGGCACGGCTAAGGGCTGCCGTTCCCCGACCAGGGCACA AGCATGCCGGTGTACGTGCAAGGCCTTTCCAAGCTGCAAAACCAGGGTGCAGACCACCAACAATCTTAGC CAGCTCTGCGGCTCTGTCATCCATTGACTATACCTATTCAAGCAGCGTGAGCGATGCGAAGTCTCGTCCC TTCCCTGCCGTCGCTCCCTTGCCATCCCCACCACGAACAGCTGACTTGGGAAACGTGTTGCCATACCTTG CCAAATTGGCCGTTGGAGAACGGCAGCTTCTATGGCGCTTCGGGGTGGCATTAATTTGCATGGTGACCTC CAAATTAGCAGGTTTGGCAGGCCCAGTACTGTTGCGTGAAGCTGTAAATGCAGTTGGCGAGCAAGCAACC GCATCCTTACGTCCTGCTGTGCATGCTGTTGTCTGCTACGGTTTATGTGGTGTCTTAGGGACTCTGGCAA AGGAGCTGCAGCACCCTACATTTGCACCTGTGTCTCAGGCTGTAGCAAGGCGTGTTGCCTACCACACTTT TGCACATGTGCTGGACTTGGATATCAAGTTTCATCTGGAGCGTAGGACTGGCCGGCTGTCGAGGATACTT GAGCGTGGTACACGTAGTGTGCAGATGCTATACCGAGCAGTGCTGTTCACCTTCATACCAACAGCATTGG AGTTTGCCTTTGTCATCGGCTTGCTGGGGACTCAGTTCAGTTCAACTGTAGCTGGACTTGTGGCAGTTAC ATTTGTAGCCTATGTGGCATGGACACTAGCAATGACACAGAGTGCTGTTGAGGTACGCAAGCAGGTCAAC ACCCTGGACAACCTCACCACCAGCAAGGCAGTAGATGCACTGTTGAATGCTGAAACAGTCACACTGTTCA ACAACCAAGCGCTGGAAGTTCAGCAGTATGACCACTACCTCAGAGGCTTTCAAAGAGCAGCAATACAGAC TGAACGCTTGAGTGCGCTTCTCAACGCTGGCCAATCTGCCATCCTAACCATTGGACTGATGCTGGTGCTG ATTGCTGCTCTTGTGAGCGCACCTGCCACTCGCCCTGTGACTGCCGGTGATTTGGTGCTGTTGCAAGGCT TGCTGCTACAGCTCTGGTCTCCTCTGCAATTCCTTGGTTGGTTTTACAGGGAGTTGAGACAGTCCTTGGT TGACATGGAGGAATTCTTTGAGATACTTCAAACCCAGAGCCAGCTGCCCGATGGCCACCTGTCATTACCC AACACGCCCCCATGCATGGTGCGAAACATTGCAGCACAAACAAGCAATAGCACAAGCCGCAATGGCAACA GCAGCCAGCGTTTGGGTAGTAACACTGTCAGCAGCACAGGCACGTCCCCACATCACAGCCACTCCCAGCA GCAGCAGCAACCGGATGATCCTACTGCCAGTTATCATGAGATACCTTATGATGCGGCATGCATCTCAGGC TTTGGGTTGGAGGTTGAACTGAAAGATGTTCACTTTGGTTATCATCCTGATAGACAAGTGCTGCGTGGTG TCACACTGCGCATACCACCCGGTCAATCAGTGGCCATCGTGGGCTCGTCAGGGTCAGGCAAATCAACCAT ACTGAAGTTAGTCACCCGGTTGTATGACGTAACCACCGGCAGCGTGGAGGTGAATGGTGTGGATATTAAA GATCTGACGAGAGATAGTTTGAGGGCAGCTGTGGCAGTTGTCCCTCAGGACACTGTGCTGTTCAATGACA CTATACTGCAAAACATCAGGTACGGTCGTCCAGAGTCAACAGATGATGAGGTCATTCGCGCAGCTCACTT GGCCCACCTCCATGATGCTGTTGTCAAGATGCCTGAGGGCTACAAGACAGTAGTGGGTGAGAGAGGTCTA AAACTCTCTGGGGGTGAGAAGCAGCGCGTTGCGATTGCGCGTGCGTTCCTGCGAGCACCACGTCTGCTGA TTTGTGATGAGGCTACGAGTGCGCTTGACAGTGCTACTGAGGCATCCATCATGAACTCACTGAATGAACT GGCTCAGGGTCGCACAAGCCTCTTTGTTGCTCACCGGCTATCCACCATCCGCAACTGTGATCGCATTGTG GTGTTATCGGCTGGTGTGGTGGTAGAGGAGGGCACACACGATCAGCTGATGTCACGTGGTGCAGTGTACA GAGACATGTGGGAGATGCAAGCAAAGGAAGCCAGTAGGGGAAATGGCGTTGCAGAGGGGACCACCTCAGA CAGTGAAGATGGGGAGCGAACAATGGAACCCCTGCCAGCTGCATTGACAGCTAAGTCCTTGAATTGA SEQ ID NO: 20 cDNA sequence starting from ATG; Cz10g28330 ATGATGCACACCTCGTCCTCAACATTGTACCCACTCGGTATTATAAGTTTCCTGGTCATTGTTGTGCCAT TAGTCATGCACGTGCTGTCTTCCCGCCGGCTGGACCTGGACCTGCTGCCCAGCCCTCCTGCTGTGCTGCT GTTGGGACACATCCAGCTGGCACTCCATGTTAAAGACATGCACCTGCAGTTTCTACGCTGGCATAACCGC TTTGGCAAGCTGTTGCGCATCAGAGTGCTGCAGCAGGATATGGTGCTCATTGCTGACCCTGCCCTGGCGT CTGAGGTGCTCACGTTGGGTCCAAACTACTGTGCACGGCGGCCAGCAGACTATGCCACCTTCAATGTGAT TCATGGCCTGTCAGCACGGCCCTCCATCCTTACTCATCAAGATGAGGCATGGTGGAAGGCTGTCAGGAGA GCAATAGCACCCGCCTACACACCTGCTGCCACCAGGGAGTTGCACAGCCTCATGGTATCCACCATGGCTC GTGTCTGTGAGCGTATCAATGCCAACCTGCACAGTGCTGCCAACAACGGCAATAGCACTAGCACCAGCAG CAGCGATAGCACTGGTAGTCCAGCAGGTATACGCATGGATGAGGAGGTGCTGGTAGCTGTGCTGGAGATC CTGTTGCATGGTTCACTGCGGTTACCACCACAAGCCATGTCATCATCAGATGTGCGGCGCACTGCACAGG TGGCCCCCAAGTTAGTGACCATTGCCAACAGCTTTGTGTCACTGCCAGGCAAGCAGTGGGTGTACACCAC ATTGCCTTTCATTTGCAAGGAAGCTCGTGTCATGCATCAGGGCCGAACCCACATGACTAATATCACACGT CAAATCTATAATCACATCAGCGCCAAATACACTGTGAATGGTGTACCGTCTGCAAAGAGTGATGTGGGAG GCTCTGAAAAGAGTGACACATCTCTCGCCTCCTGCTTGATGCGCCTGGCCCACCCTCAACACACGCCTGG TCAAACATTCACCCAGGATGATATCATGGCTGAGATTGCCATCAACATCATAGGACAGGGATCCGTACCA TGGACAGTATCTTGGGCACTGTTCCAGCTGACTCAGCGGCCTGATGTGGAGAGCCGATTGCTGTCAGAGC TACAGGGCCTGGGGTTACCCTGTGATGGTGACCTGGCGGCAGCATGTGATGCCATTAGTCATCCGGAGTG TCTCAGGGACACCCCCTACCTGACAGCTGTCATTAATGAGGTGATGCGCATGTACCCAGCTGGTGTGTCA GCAGCTCCTAGGTTGACAGAACAGCCCATCAAGCTTGGAGGGTATCGCATACCTGCTGGCGTTATGGTCT TCCCAAATTTATTCACCATCATGAATTATAGAGGTAACTGGCAGCAGCCAGATCAGTTTTACCCAGATCG CTGGCTGCAACCAAACTCATCAGTAGACCCCTCTTCAGGCGCCCCAAGGTTTATCCCCTTCAGCATCGGG CCAAAGATTTGCATAGCACAACATCTGGCCATAATGCAGTGTAAGATTATGTTGACATTGTTAGTCAGCT GCTTGAAATTCCAGCTGGCACCCACAATGGGGGGAATTGAGGGGGTGATGAAACGGGTAGAGGCGACGTT GGAGTTGCGGGTGCAGGGGGGCTTGTGGTTTACTGCAGAGATCAGGAGGTCTAACAATGTAAAATGA SEQ ID NO: 21 cDNA sequence starting from ATG; Cz11g14160 ATGGATGGACTTGCGATCGCCATGGGCTGTGCCGCGGCGGTGCTGGTGTGCTATACGGTGTTGCCATTCC TGTGCAGCCTTGCACTAGCCATCTGGAGGTTTCATACAAGCAAGATACCCGGGCCTCCAGCAAAAGACTT CATCCTGGGCCATGCTGCGGCTCACTTGAATGACAAGGCTCCTTTTATTTTCAAAAGATGGGCTGAGAAA TATGGCAAACTTTACAAGGTTCGGATGGCCGACAACTTCGCCGTGGTGCTGACCGATCCCGAAGTCATAC AGTCCATAGCTCGCAAAATACAAAAGCCTACCAAATCGTACAGTAGTCTTGAGCTGGGCACGTTCCCCAA GGATCACAACATCTTGACCGCTCCAGATGGCCCACACTGGAAAGCAGTAAGACAAGGCATAACACCAGCA TTCAGTGTAGCAAACTTGAAGCAGGTATTCCCCTGGTTGTGCCATGTCACCAAGGTGGCTGCAGCTAAAC TCCAGGACACAGGCCCTTCCACCAGCTTTGACATTTCTGACATTGCCAAGCGGATCACCAGTGATGTCAT TGGGCAGCTGCTGTATGCGGAAGACCTGGGAGCCATAGCATATAGGCCCAGTGAATACCTGGAGCTGTTC CAGATCGGCATCACTGCAGCTCATAAGAGCCTTGGGAGACCTTTACGGCTGTATGCCATCTGGGATCCTG AGGTGAGGCGTCAGAACCGCGCAATAAACCGTCTTGACCAGTAG 

What is claimed is:
 1. An expression vector comprising a nucleic acid sequence that encodes a polypeptide involved in astaxanthin production, wherein the polypeptide has at least 90% identity to any one of SEQ ID NOS: 1 to
 7. 2. The expression vector of claim 1, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:1 to
 7. 3. The expression vector of claim 1, wherein the nucleic acid sequence is a cDNA or genomic DNA sequence from a Chloroformis zofingiensis gene Cz04g21110, Cz05g17060, Cz09g27180, Cz08g16130, Cz10g28330, Cz11g14160, Cz02g29020.
 4. The expression vector of claim 1, wherein the nucleic acid sequence encodes an ABC transporter.
 5. The expression vector of claim 4, wherein the nucleic acid sequence is a cDNA or genomic sequence from a Chloroformis zofingiensis gene Cz04g21110, Cz05g17060, Cz09g27180, or Cz08g16130.
 6. The expression vector of claim 1, wherein the nucleic acid sequence encodes a cytochrome P450 protein.
 7. The expression vector of claim 6, wherein the nucleic acid sequence is a cDNA or genomic sequence from a Chloroformis zofingiensis gene Cz10g28330 or Cz11g14160.
 8. The expression vector of claim 1, wherein the nucleic acid encodes an acyltransferase.
 9. The expression vector of claim 8, wherein the nucleic acid sequence is a cDNA or genomic sequence from a Chloroformis zofingiensis gene Cz02g29020.
 10. The expression vector of claim 1, wherein the nucleic acid sequence has at least 70% identity to any one of SEQ ID NOS:8 to
 21. 11. The expression vector of claim 1, wherein the nucleic acid sequence has at least 70% identity to any one of SEQ ID NOS:15 to
 21. 12. The expression vector of claim 1, wherein the nucleic acid sequence has at least 90% identity to any one of SEQ ID NOS:15 to
 21. 13. The expression vector of claim 1, wherein the nucleic acid sequence comprises a sequence of any one of SEQ ID NOS:15 to
 21. 14. A host cell comprising expression vector of claim
 1. 15. A host cell comprising a heterologous nuclieic acid sequence encoding at least one astaxanthin polypeptide polypeptides, wherein the at least one polypeptide has at least 90% identity to any one of SEQ ID NOS: 1 to
 7. 16. The host cell of claim 15, wherein the at least one polypeptide comprises the amino acid sequence of any one of SEQ ID NOS: 1 to
 7. 17. The host cell of claim 15, comprising a heterologous nucleic acid encoding one, two, three, four, five, six, or seven of SEQ ID NOS: 1 to
 7. 18. A method of producing astaxanthin, the method comprising culturing a host cell of claim 14 under conditions in which astaxanthin is produced.
 19. An isolated nucleic acid comprising a cDNA sequence of any one of SEQ ID NOS:15 to
 21. 